COLORECTAL CANCER FAMILY
REGISTRY
UNIVERSITY
OF SOUTHERN CALIFORNIA
NORRIS CANCER CENTER
1
COLORECTAL CANCER FAMILY REGISTRY
UNIVERSITY OF SOUTHERN CALIFORNIA
NORRIS CANCER CENTER
__________________________________________
_
PROTOCOL MANUAL for
HANDLING OF BIOLOGICAL SPECIMENS
BIOSPECIMEN PROCESSING
Banking Flow Chart
Biospecimen Availability
A. Peripheral blood
Flow Chart
About blood collection
Guideline for receiving blood
After the blood arrives
Summary
1. Blood Spot Preparation
2. Plasma and Buffy Coat Separation
3. Ficoll Separation
B. Frozen tissue
C. Paraffin-embedded tissue
D. Others: Buccal Smears, etc.
 Supplies
FINAL CELLULAR AND GENETIC MATERIAL PREPARATION
A. DNA
1. from fresh whole blood
2. from buffy coat
3. from dry blood stains
4. from frozen tissue
5. from paraffin-embedded tissue
6. from others (plasma, urine, buccal smear...)
B. RNA
1. from fresh whole blood
2. from buffy coat
3. from cells
C. Lymphoblastoid cell line
 Supplies
Colorectal Cancer Family Registry - Coordinating Center Laboratory
Drafted October 10, 1998
2
University of Southern California, Department of Preventive Medicine
Revised Nov 23, 1998 AD
3
STORAGE, PACKING AND SHIPPING
A. Storage
Location of all storage
Preventive maintenance
B. Packing and shipping
 Supplies
SPECIMEN DATABASE
A. Scheme for specimen ID assignment
B. Scheme for location assignment of biospecimens
C. Biospecimen labeling guideline
D. Biospecimen data records and report
E. Biospecimen database tables
 Supplies
QUALITY CONTROL
Summary of Routine QC for all specimens
A. Routine QC for DNA
B. Routine QC for RNA
C. Routine QC for Cell Cultures
D. Special QC for DNA
E. QC Exchange Plan
General Protocol for Lab QC and QA
 Supplies
STANDARDIZED LAB WORKSHEETS
USC COORDINATING LABORATORY SETUP
A. Resource and Environment
B. Safety Requirement
1. Biosafety
2. Radiation safety
C. Contamination Control
1. For PCR-based experiment
2. For RNA-based experiment
3. For tissue culture work
D. USC Safety Resource
REFERENCES
Colorectal Cancer Family Registry - Coordinating Center Laboratory
University of Southern California, Department of Preventive Medicine
Drafted October 10, 1998
Revised Nov 23, 1998 AD
4
BIOSPECIMEN PROCESSING
5
Biological Specimen Banking
for the
COLORECTAL CANCER FAMILY REGISTRY
STUDY SUBJECTS
DNA back up source:
Buccal swabs...
Peripheral Blood
Archival specimens
Paraffin blocks
or
slides
EDTA, purple
Blood spots
Store at
room
temperature
DNA
ACD, yellow
Centrifugation
Plasma
Buffy Coat
preparation
preparation
Ficoll separation
of
white blood cells
Store in liquid
nitrogen
Lymphoblastoid
Cell Lines
Freeze at
-70 degrees
centigrade
PLASMA
Colorectal Cancer Family Registry - Coordinating Center Laboratory
Uni versity of Southern California, Department of Preventive Medicine
Freeze at
-70 degrees
centigrade
DNA
and
DNA
RNA
Drafted November 6, 1997
Revised October 2, 2002
6
CFR
Biospecimen Availability
(Draft modified from CFRBCS and CFRCCS)
Biospecimen products available to researchers from CFR participants
Product
Original Amount
Aliquots
Storage Condition
Available to
Comments
researchers?
Plasma
10-12mL
18-24
-70C
Yes
Nonrenewable product
Room temperature
No
Nonrenewable;
@ 0.5mL each
Blood Spots
DNA
44 spots of 20ul
2 cards of 22 spots
each
each
0.4-0.5mg from
Upon request
For QC and back up
-20C
Yes
EDTA
Preferred product for distribution;
Inexpensive
more from EBV
Ficoll-separated WBCs from 10mL blood
~4 aliquots
Liquid N2
No
@ 2x106 cells each
EBV-transformed WBC
Theoretically
NA
Source of EBV lines and non-EBV RNA;
Nonrenewable product
Liquid N2
Yes
unlimited
Provides DNA and RNA;
Only renewable product in CFR.
H&E stained paraffin-
2 slides per block (possible
2 slides @ 5
embedded tissue
multi-blocks per case)
um thickness
Paraffin-embedded
10 sections per block
10 slides @ 5 um
tissue on ProbeOn
(possible multi-blocks
thickness
Plus slides
per case)
Paraffin-embedded
16-26 sections per
16-26 slides @
tissue on regular slides
block (possible multi-
5 um thickness
Room temperature
No
Nonrenewable;
Upon request
Room temperature
Yes
Nonrenewable;
For immunohistochemical analyses
Room temperature
Yes
Nonrenewable;
Provides DNA.
blocks per case)
Paraffin-embedded
4 sections per block
4 sections @ 5 um
Room
tissue in 1.5 ml
(possible multi-
thickness in 4 1.5- ml tubes
temperature
microcentrifuge tubes
blocks per case)
unmounted on any slides
Colorectal Cancer Family Registry - Coordinating Center Laboratory
University of Southern California, Department of Preventive Medicine
Yes
Nonrenewable;
Provides DNA.
Drafted October 10, 1998
Revised December 30, 2002
7
A. Processing the peripheral blood
8
PROCESSING THE BLOOD SAMPLES
Receive three tubes of EDTA blood
(3 X 10 ml purple tops)
and one ACD tubes
(1 X 8.5 ml yellow tops)
3 X 10 ml EDTA tubes
24-30 ml only
23-29 ml from
EDTA tubes
1 ml from
EDTA tubes
1 X 8.5 ml
ACD tubes
Removes
granulocytes
and 2/3 of DNA
Consider
reclamation of
granulocytes
for DNA/RNA
Ficoll separate
WBC
22 Blood
spots of 20 ul
~12 ml
plasma
Aliquot in
0.5 ml tubes
Freeze at
-700C
Buffy coat
Aliquot in 1.5 ml tubes
Cryo 4 x 2x106 cell
aliquots in liquid
N2
Extract DNA now
or freeze buffy coat
at -700C
EBV lines can
be used as
inexhaustible
sources of both
RNA and DNA
For future EBV
transforming
5 micrograms DNA/ 1 million WBCs
1 ml whole blood = 5 million WBCs
9 ml whole blood = 225 micrograms of DNA = 225,000 nanograms
Usual PCR reaction takes 25 nanograms > 9000 reactions / 9 ml of blood
T75 flask contains only 3-5 million cells
CFRBCS uses [50 ug/ml] DNA ( Feb 25, 1997 minutes)
9cc of blood resulting in 6cc of plasma ( June 11, 1998 minutes)
Anh’s revision 10-02-02
Colorectal Cancer Family Registry - Coordinating Center Laboratory
University of Southern California, Department of Preventive Medicine
Drafted October 10, 1998
Revised Nov 23, 1998 AD
9
About blood collection
Blood will be drawn from Sunday to Thursday and shipped from Monday to
Thursday. Please allow 72 hours notice for shipments that occur under special
circumstances, such as shipments that will arrive on a weekend or a holiday. Any blood
not received by the lab within 24 hours of it being drawn will be rendered unusable for
ficoll separation. In this case, the blood will be used for plasma and buffy coat. All blood
should be shipped at room temperature using the “Two-Layer Insulation Method”. If the
temperature of the blood exceeds room temperature during shipping, ice packs should be
used. The blood should never be in direct contact with these ice packs. If drawing an
amount below the minimal request, a second draw should be set up, if feasible. Once
received, the blood should be kept at room temperature until processed. All samples sent
to the lab will use study ID numbers only.
Priority of collecting blood samples
The goal of the phlebotomy visit is to collect 4 tubes of blood in the following order:
-1 ACD tube (~8.5cc blood each): Shipped to Coriell by USC after ficoll separation
for future establishment of cell lines.
-3 EDTA tubes (~7.5cc to 10cc blood each) Shipped to USC lab to be used for DNA
extraction.
 When conducting the blood draw, always begin with filling the EDTA tubes.
 Only after all 3 EDTA tubes are filled should the ACD tube be filled.
 In all cases, filling of the EDTA tubes take precedence.
Filling the blood collection tubes
Tubes should be filled to the indicated, colored line on the manufacturer’s label, which is
about ¼” below the stopper.
Time between blood draw and arrival at lab
The goal is to have blood samples arrive at the lab within 24 hours of the blood draw.
Any blood not received by the lab within 24 hours of being drawn will be used for DNA
isolation only, not for plasma. If the time between the blood draw and shipping is
delayed, so that more than 24 hours elapse between draw and processing at the lab, a
second draw should be requested if feasible.
No samples are ever to be discarded even in a blood drawing error!
Colorectal Cancer Family Registry - Coordinating Center Laboratory
University of Southern California, Department of Preventive Medicine
Drafted October 10, 1998
Revised Dec 30, 2002 AD
10
Guideline for receiving blood
Between Coordinating Centers and Processing Lab in USC






As soon as a blood draw is scheduled, please inform immediately all lab staffs who
work for this study (give at least 72 hours advance notice). They will primarily
oversee and process the biological specimen for the registry.
Once the blood is sent out, inform the lab the date and the number of subjects of the
shipment.
If the lab is to expect the shipment on a weekend or a holiday, please also include
the tracking number of the shipment and sent directly to the lab.
If no confirmation is returned from the lab about the receiving schedule, please find to
inform them in person.
After the shipment arrives, bring it to the lab at room 5427 where a section of Thanh’s
bench is designated to receive samples for the registry. Unpack and leave the blood
tubes standing upright in the assigned rack. Information such as the subject’s study
ID, date and time (Pacific time) drawn must be provided.
Finally, the lab should be informed promptly if there is any cancellation.
Anh Diep
Ehab El-Khouly
Thanh Diep
Evgenia Ter-Karapetova
e-mail address
atdiep@hsc.usc.edu
elkhouly@hsc.usc.edu
thanhtdi@hsc.usc.edu
terkarap@hsc.usc.edu
work number
(323) 865-0713
(323) 865-0582
(323) 865-0583
(323) 865-0714
emergency contact
(818) 361-7503
(818) 265-9431
(818) 365-8095
(626) 296-2766
Lab address
(when a shipment is to be sent on a weekend or a holiday)
Anh T. Diep c/o Haile’s Lab
USC SCH of MED-Norris Cancer Center
Topping Tower 5427, 5429
1441 Eastlake Ave.
Los Angeles, CA 90033
Lab Telephone Line #1: (323) 865-0585
Lab Telephone Line #2: (323) 865-0586
Colorectal Cancer Family Registry - Coordinating Center Laboratory
University of Southern California, Department of Preventive Medicine
Drafted October 10, 1998
Revised July 8, 1999 AD
11
Blood Delivery Notification Procedure
1.
As soon as a blood draw is scheduled, please inform the following people:
Email address
atdiep@hsc.usc.edu
Anh Diep
Evgenia Ter-Karapetova terkarap@hsc.usc.edu
thanhtdi@hsc.usc.edu
Thanh Diep
2.
Work number
(323) 865-0713
(323) 865-0714
(323) 865-0583
Emergency contact
(818) 361-7503
(626) 296-2766
(661) 799-9726
Ship the blood directly to our processing lab in USC. The attention line and the
address are:
Anh Diep c/o Haile’s Lab
USC Norris Cancer Center
1441 Eastlake Ave, NOR 5427
Los Angeles, CA 90033
3.
The labels for blood tubes should be preprinted with these information:
Study ID
Time Drawn
Initial of phlebotomist
Date Drawn
Time Zone
(EST, CST, PST, ...)
Stick the labels on top of the writing area of the tube.
4.
Once the blood draw is confirmed, email all the above three personnel using the
following format:
Name of Center.........................
Study ID....................................
Drawn In...................................(name of state the blood was drawn in)
Date Drawn...............................
FedEx Tracking #......................
Colorectal Cancer Family Registry - Coordinating Center Laboratory
University of Southern California, Department of Preventive Medicine
Drafted July 11, 1999 TD
Revised July 8, 1999 AD
12
Special instructions for shipments that will arrive on a weekend

Please provide 72 hours notice for shipments that will arrive on a weekend.

Please follow the special instructions for these types of deliveries:


When filling out the FedEx forms for weekend delivery of bloods, you must:
1. Sign the air bill so shipments can be dropped off even without obtaining a
signature from the lab.
2. Write a note for FedEx (next to our address on the forms) which says:
"Please do not deliver before 10:30 am (PST)."
It is imperative that this is done because FedEx only makes one delivery attempt for a
Saturday delivery, and we can't afford to miss it.
USC observes 14 holidays in 2001 - please do not schedule bloods to arrive at USC
on these days:
New Year’s Day
Martin Luther King, Jr. Day
President’s Day
Memorial Day Observed
Independence Day
Labor Day
Thanksgiving Day
Day after Thanksgiving Day
Christmas Eve
Christmas
Between Christmas and New Year’s Day
New Year’s Eve (observed)
Colorectal Cancer Family Registry - Coordinating Center Laboratory
University of Southern California, Department of Preventive Medicine
Monday, January 1, 2001
Monday, January 15, 2001
Monday, February 19, 2001
Monday, May 28, 2001
Wednesday, July 4, 2001
Monday, September 3, 2001
Thursday, November 22, 2001
Friday, November 23, 2001
Monday, December 24, 2001
Tuesday, December 25, 2001
December 26, 27 and 28, 2001
Monday, December 31, 2001
Drafted July 11, 1999 TD
Revised July 8, 1999 AD
13
Labeling blood collection tubes
Each blood collection tube must be labeled with the following information:
Study ID
Date of draw,
Time of draw and time zone (see Time Zone Table)
Phlebotomist’s initials
Study identifier: CFR
The label must be placed vertically along the length of the tube covering the writing area
of the tube so that lab will be able to view the level of the blood in the tube. Avery
Labels Style 5667 (0.5” x 1.75”, height x length) is suggested for use.
All samples sent to the lab will be labeled with study ID numbers only.
Do not include any personal identifiers such as name or address.
If possible, blood tube labels should be preprinted at the data collection centers using the
Excel blood collection tube label generator.
Time Drawn_____________EDT
These labels include the Study ID and
corresponding bar code, date of draw, time of
Phlebotomist: 1234
CFR
draw and time zone (see Time Zone Table),
phlebotomist’s ID, and the study identifier, CFR.
*111111*
01/02/01
In cases where the phlebotomist or the date of draw is unknown or may change, leave the
respective fields blank in the label generator and the label shown at left will be generated:
The date of draw and phlebotomist ID can be handwritten in just prior to the blood draw.
US Time Zone Table
The following table contains time zones in Standard and Daylight Savings Time for each
center’s registry catchment’s areas. If the blood draw is done by a physician or outside
phlebotomy service in another time zone, then that time zone should be recorded on the
tube label.
US Center
Arizona
Cleveland
Minnesota
North Carolina
Colorado
Dartmouth
USC
Standard Time
Through 3/31/01
10/28/01-4/6/02
10/27/02-4/5/03
PST
EST
CST
EST
MST
PST
PST
Colorectal Cancer Family Registry - Coordinating Center Laboratory
University of Southern California, Department of Preventive Medicine
Daylight Savings Time
4/1/01-10/27/01
4/7/02-10/26/02
4/6/03-10/25/04
PDT
EDT
CDT
EDT
MDT
PDT
PDT
Drafted October 10, 1998
Revised Nov 22, 2001 AD
14
Packaging the blood collection tubes for shipment
“Two-layer insulation packing method”
Supplies required:
5-tube foam carrier
Ziploc bags
Insulated foam carton
Cardboard box
Absorbent wadding material
Tape for sealing boxes
Biohazard label
Express shipping air bill
1.
2.
3.
4.
5.
6.
Blood collection tubes are placed in the 5-tube foam carrier, which is then
taped shut.
The sealed 5-tube foam carrier is placed in a Ziploc bag with absorbent
material.
The 5-tube carrier is then placed in the large insulated box with additional
absorbent material.
The foam box is securely taped shut and placed in the cardboard outer box.
Affix a bright orange biohazard label to the cardboard outer box.
The cardboard box is then securely taped shut and the completed air bill is
affixed.
Blood shipment temperature
All blood should be shipped at room temperature using the two-layer insulation packing
method. This system plus sufficient taping is the best blood shipment method. Do not
include ice packs when using this system.
Colorectal Cancer Family Registry - Coordinating Center Laboratory
University of Southern California, Department of Preventive Medicine
Drafted October 10, 1998
Revised Nov 22, 2001 AD
15
After the blood arrives
At the USC Coordinating Center Laboratory
During the initial founding of the registry, the USC laboratory staff will be
archiving blood samples to facilitate future analysis. No analysis will be done at this
point. However, by collecting a maximum amount of blood and through careful storage,
we will create a potentially inexhaustible resource.
Participants are asked to donate less than 40cc. Three EDTA tubes and one ACD
tube will be processed differently, so that the EDTA tubes become a source of plasma and
buffy coat, and the ACD tubes become a source of white blood cells.
Processing the EDTA tubes
From the 16cc to 20cc of blood in the EDTA tubes, 1cc in homogeneous phase
will be drawn immediately to make 44 blood spots on filter paper, which will be stored at
room temperature. This will serve both as a back-up source of DNA as well as a quality
control source for DNA.
Our data from previous studies have shown that a 3mm square (from 3ul of whole blood)
of dried bloodstain provides enough DNA for 2 to 5 PCR reactions. Thus, 44 blood spots
theoretically can yield sufficient DNA for about 1000 PCR reactions.
The remaining 15cc to 19cc of whole blood from these two EDTA tubes are
separated by centrifugation and aliquoted into 0.5ml tubes of plasma and 1.0ml tubes of
buffy coat. Separation needs to be done within 48 hours after the blood was drawn. The
plasma and the buffy coat will each be saved separately and frozen at -70C. The plasma
will be used for later phenotypic studies, and the buffy coat will serve as a source of DNA
once funding is available for DNA extraction.
The amount of plasma collectible from 15-19ml blood is 10-12ml. The amount of buffy
coat from 15cc to 19cc whole blood should provide enough DNA for 15,000 PCR
reactions (20-25ng DNA template). Genotyping of any marker takes about 50ng DNA as
a template, which is enough for 2 PCR reactions. Thus, the DNA allows us to genotype
more than 7000 markers. Also, to characterize genes that are already cloned and
identified as causes of colorectal cancer by screening the entire gene to assess
presence/absence of a mutation, our current work suggests we will need a minimum of 25 ug DNA or 1-5 cc of blood for 8-12 kb in length. Thus, the DNA allows us to screen a
number of genes.
Colorectal Cancer Family Registry - Coordinating Center Laboratory
University of Southern California, Department of Preventive Medicine
Drafted October 10, 1998
Revised Nov 23, 1998 AD
16
Processing the ACD tubes
For the one ACD tube of about 10cc whole blood, we will separate the white
blood cells through Ficoll gradient columns. These cells will be stored at -70C for future
lymphoblastoid cell line establishment. Culturing these cells would provide us with an
inexhaustible source of RNA and DNA. 10cc whole blood requested from the subjects
after Ficoll separation will provide 4 ampules of white blood cells. We will cryopreserve
in liquid nitrogen for EBV transformation.
Colorectal Cancer Family Registry - Coordinating Center Laboratory
University of Southern California, Department of Preventive Medicine
Drafted October 10, 1998
Revised Dec 30, 2002 AD
17
Summary
Aliquots made from >20 ml EDTA blood into:
2 cards of 44 blood spots (22 per card, 20 ul each spot) for direct PCR
20 to 24 aliquots (0.5 ml each) of plasma
3 to 5 buffy coat aliquots (0.75 ml each) for DNA isolation
at room temp
in -70oC
in -70oC
Aliquots from 10 ml ACD blood into:
4 aliquots (1 ml) of white blood cells
in LN
1-15 ml tube of residual cells from both EDTA and ACD tubes for more DNA collection
in -70oC
If less than 20 ml EDTA blood is received:
Always reserve 2 cards of blood spots regardless of the volume of blood received.
Make less blood pellets for DNA isolation.
Make less aliquots of plasma for phenotyping.
If less than 10 ml of EDTA blood is received, a second draw should be setup if feasible.
If less than 5 ml ACD blood is received:
Make less aliquots of white blood cells.
If less than 5 ml of ACD blood is received, a second draw should be setup if feasible.
If only ACD blood is received and second draw is not feasible:
Since ACD tubes will be drawn first.
Always reserve 2 cards of blood spots.
Regardless of the volume of blood or the type of blood tube received.
Make blood pellets for DNA isolation from this only one ACD tube (<8.5mL blood).
Make as many aliquots of plasma as possible from that one ACD tube (<5.0mL plasma).
Make note for this “no ficoll seperated WBCs” specimen.
If blood cannot be processed within 48 hours of it being drawn, and second draw is not
feasible:
Reserve 2 cards of blood spots as always from EDTA tubes.
Make blood pellets for DNA isolation from EDTA tubes.
Make as many aliquots of plasma as possible from EDTA tubes.
There will be no aliquot of white blood cells for EBV cell line.
Make note for this “ no ficoll seperated WBCs” specimen.
Colorectal Cancer Family Registry - Coordinating Center Laboratory
University of Southern California, Department of Preventive Medicine
Drafted October 10, 1998
Revised Dec 30, 2002 AD
18
1. Blood Spot Preparation
(Modification of Brockmoeller et al. and Lin 1992 )
Dot on filter paper 44 spots from 1 ml whole blood. Reserve for quality control and a
DNA back up source.
Reagents and Equipment:
75% alcohol
aerosol resistant tips
balance
banker box
Betadine
biohazard waste containers: bench top and step can
bleach
blue under pad
dotting grid
envelopes
gloves
kimwipes
P20 pipetmen
rack for 15 ml tubes
S&S 903 filter papers*
stop removers
Blood protective pads
TainerTop Safety Closure
wax paper
Stepwise Procedure:
a. Mix blood thoroughly by gentle inversion to have a homogeneous phase before
starting. Blood drawn in purple top vacutainer (EDTA as the anticoagulant).
b. Wipe top of vacutainers with ethanol before opening the cap. Open the blood tubes
with stop removers.
c. Pipet 20 ul of blood and dot onto the paper. Recap blood drawing tubes with
TainerTop Safety Closure during processing to prevent cross-contamination.
d. Make two full blood cards of 22 spots each.
e. Airdry thoroughly.
f. Store in paper envelopes at room temperature.
*Sharing from N. Lindor: Guthrie Spots: One "pearl" that I was able to glean from Schliecher and Schuell is
that of circumspect in regards to printing circles for blood spots. S&S uses only inert inks, and printing
presses that are used expressly for that purpose. In contrast, having S&S paper printed at a local printer,
while saving money, may compromise the DNA extraction process. Only biologically inert inks, such as
soy-based, should be used, and even then the ink may be contaminated by ink used in the previous printing
job.
Colorectal Cancer Family Registry - Coordinating Center Laboratory
University of Southern California, Department of Preventive Medicine
Drafted October 10, 1998
Revised Nov. 26, 2001 AD
19
2. Plasma and Buffy Coat Separation
(Gustafson et al.1987)
Centrifuge at 1300-1500g for 15 to 30 minutes. Pipet out the supernatant plasma
with a transfer pipette. The plasma is the yellowish hue top layer. Then with
another transfer pipette, carefully transfer the buffy coat to fresh tubes. The buffy
coat is a broad band of white blood cells with heterogeneous density. Store both this
plasma and buffy coat for further application.
Reagents and Equipment:
0.5 ml screw cap microcentrfuge tubes
2.0 ml screw cap microcentrfuge tubes
1 x Hanks’ Balanced Salt Solution
balance
Betadine
biohazard waste containers: bench top and step can
bleach
blue under pad
gloves
glycerol freezing solution
horizontal rotor 221
IEC HN-SII bench top centrifuge
kimwipes
racks for 15 ml centrfuge tubes and microcentrfuge tubes
rubber adaptors
transfer pipets
storage box
Stepwise Procedure:
a. Mix the fresh whole blood by gentle inversion.
Blood drawn in purple top vacutainer (EDTA as the anticoagulant).
Recap blood drawing tubes during processing with TainerTop Safety Closure to
prevent cross contamination.
b. Centrifuge vacutainers at 1500xg for 30 min to separate plasma from others.
Make sure tubes are balanced before centrifuging.
Make sure tubes are recapped tightly.
Use IEC HN-SII bench model centrifuge, horizontal rotor 221; Can hold 6x15 ml
centrifuge tubes. Set speed at 3/4, about 2000-3000 rpm for 1500xg.
Colorectal Cancer Family Registry - Coordinating Center Laboratory
University of Southern California, Department of Preventive Medicine
Drafted October 10, 1998
Revised Nov 12, 1998 AD
20
c. Remove plasma with transfer pipets to 0.5 ml screw cap microtubes.
The sample should have three distinct layers:
i, Plasma (yellowish hue)--Top layer.
ii, Buffy coat (white)-- thin interface.
iii, Red blood cells---bottom layer.
d. Fill tubes to 0.5 ml only and make as many aliquots as possible.
e. Carefully pipet out the dense interface of buffy coat with a transfer pipet into 2.0 ml
screw cap microtubes.
f. Fill tubes to 0.75 ml only and also make as many aliquots as possible.
It is acceptable to have some red cells mix with buffy coat. Efforts to avoid red cells
can lead to considerable loss of white cells and diminished DNA/RNA yield.
g. Overlay the white cells with the glycerol freezing solution (GFS) to 1:1 blood to GFS
(with a maximum of 1.5 ml final volume). Gently mix by inversion.
Glycerol freezing solution( pH 7.4 )
Reagent
MW (g)
Amount per 0.5 L
Final concentration (mmol/ L)
Citric acid, sodium salt
294
7.35g
50
Sodium phosphate, monobasic,
138
1.38g
20
monohydrate NaH2PO4-H2O
Sodium phosphate, dibasic, anhydrous
142
1.32g
20
NaH2PO4
99% glycerol
200mL
Filter sterilization using 0.2 u cellulose acetate or hydrophillic polyethersulfone low binding protein membrane
h. Place the buffy coat tubes into a rate-control freezing container and freeze in -70C
freezer.
i. Transfer residual cells from remaining blood to a 15 ml centrifuge tube.
j. Raise the volume of the 15 ml tube to 7.5 ml with GFS for more DNA collection.
k. Store all tubes in -70oC immediately.
20-24 0.5 ml micro tubes of plasma, 3-4 2.0 ml micro tubes of buffy coat and one
15ml centrifuge tube of residual cells.
Colorectal Cancer Family Registry - Coordinating Center Laboratory
University of Southern California, Department of Preventive Medicine
Drafted October 10, 1998
Revised Feb. 14, 2000 AD
21
3. Ficoll Separation
(modified from NYCOMED PHARMA AS Diagnostics Lymphoprep protocol)
Reagents and Equipment:
Alcohol, 70%--in a spray bottle
DMSO (Dimethyl Sulfoxide)
FBS (Fetal Bovine Serum)
HBSS (Hanks' Balanced Salt Solution), 1X
Lymphoprep (from Life Technologies)
RPMI Medium 1640, 1X
Trypan Blue Stain, 0.4%
Adapters, for rotor, each accommodates 8 of 15 mL tubes
Adapters, for rotor, each accommodates 3 of 50 mL tubes
Beaker containing bleach for blood waste
BloodBloc Pads
Burner
Centrifuge tubes, 15 mL
Centrifuge tubes, 50 mL
Cryogenic vials, 1.2 mL
Hemacytometer
Hood
Sterilized with 75% alcohol and UV shined for half an hour before and
after every procedure
Microscope
Microtubes, 0.5 mL
Pipet-Aid
Pipetman and aerosol barrier tips, 20 ul
Pipetman and tips, 200ul
Serological pipets, 1 mL
Serological pipets, 2 mL
Serological pipets, 5 mL
Serological pipets, 10 mL
Serological pipets, 25 mL
Sterilized transfer pipets
Centrifuge--Sorvall RC 26 Plus
Rotor, swing-out bucket—SH-3000, hold 4 buckets
Stepwise Procedure
a. Record down total blood volume (subtract 1.5 mL of ACD solution from each
vaccutainer). Dilute the blood with HBSS (1:1) and mix well.
Blood drawn in yellow top vacutainer (ACD as the anticoagulant)
b. Carefully overlay the diluted blood over Lymphoprep (the volume of
Lymphoprep is half the volume of the diluted blood). Avoid mixing.
Colorectal Cancer Family Registry - Coordinating Center Laboratory
University of Southern California, Department of Preventive Medicine
Drafted October 10, 1998
Revised on July 14, 1999 by TD
22
c. Centrifuge at 2000 rpm, 18-22C for 30 min with BRAKE OFF.
d. Discard most of the top layer (plasma) without disturbing the next layer that
contains lymphocyte.
e. Remove the lymphocyte layer to a new centrifuge tube.
Avoid picking up ficoll.
f. Wash the harvested lymphocyte with HBSS. Mix well.
g. Centrifuge at 1000 rpm for 10 min.
h. Discard the supernatant.
i. Resuspend the pellets with 13 mL of RPMI. Mix well. Save 10L of this solution
in a microtube for cell counting purpose. Centrifuge the rest at 1000 rpm for 10
min.
j. Discard the supernatant.
k. Suspend the cells from every 2 mL of blood in 1 mL of freezing media.
(63.5%RPMI + 30%FBS + 6.5%DMSO).
DO NOT add DMSO until ready to freeze the cells.
l. Divide the suspended cells to the pre-labeled cryogenic vials (0.5 mL per tube).
m. Place the cryogenic vials into a rate-control freezing container and freeze in
-80C freezer.
n. Remove them to the cryogenic container on a weekly basis.
o. Rinse vacutainers (both ACD and EDTA) and tubes with 1x HBSS and combine
all in a 15 mL centrifuge tube to save the residual cells for more DNA collection.
p. Prepare counting solution: Add 190 L of Trypan Blue to the microtube with 10
L of the cell suspension solution (20 x dilution). Mix well.
q. Count the cells.
Keep separate counts of healthy and dead cells.
r. Calculations:
Total number of cells yielded = X= Total counts / 2 / 4 * 20 * 10000 * 13
Lymphocyte Counts per ml of blood = X / Total Blood Volume
Viability = Counts of Healthy Cells / (Counts of Both Healthy + Dead Cells) *
100%
Colorectal Cancer Family Registry - Coordinating Center Laboratory
University of Southern California, Department of Preventive Medicine
Drafted October 10, 1998
Revised on July 14, 1999 by TD
23
B. Processing the frozen tissue
24
If frozen tissue from a participant is available, it has to be frozen in liquid nitrogen
immediately after surgery and sent to the USC biospecimen-processing lab for further
processing.
Colorectal Cancer Family Registry - Coordinating Center Laboratory
University of Southern California, Department of Preventive Medicine
Drafted October 10, 1998
Revised _______________
25
C.
Processing of
Paraffin-embedded tissue
USC PHASE III
Colorectal Cancer Family Registry - Coordinating Center Laboratory
University of Southern California, Department of Preventive Medicine
Drafted October 10, 1998
Revised Jan 29, 2004 AD
26





Information needed for selecting PETs:
 For TMA
 CCF Only
Accessing the paraffin-embedded-tissue block/slides
a. Tracking
b. Accessing
Protocol for paraffin-embedded-tissue section preparation (For CCF Only)
 Reagents and Equipment
A. Order of the paraffin-embedded-tissue sections
B. Preparation of the paraffin-embedded-tissue sections
The Alternative Protocol for Centers that cannot provide the complete
protocol with 42 sections
 Reagents and Equipment
A. Order of the paraffin-embedded-tissue sections
B. Preparation of the paraffin-embedded-tissue sections
Tissue Micro-array standard operating procedure(Phase II)
a. Equipment & Accessories
b. Reagents and supplies
c. Preparation of the blank recipient array block
d. Preparation of the donor blocks and slides
e. Designing and constructing the Array (with cell carry-over free instruction).
f. Align the punch
g. Complete PET processing protocol and the order of the PET banking procedure
h. After the construction of a full array block is completed and before sectioning
i. Sectioning the blocks (donor and array)
j. Tape-sectioning the array block 0.6 mm in diameter and spacing between two
k. Hematoxylin And Eosin Staining Procedure

Specimen, Reagents, Stepwise, Result, References, Troubleshooting
l. Instruction to use the four block indexer
m. Instruction to use the depth stop kit
n. Maintenance of the Tissue Arrayer
o. Worksheets
p. Barcode labels
q. Storage
r. Data file
s. Software for Spot Imaging Documentation (TO COME)
t. TROUBLESHOOTING guide for manual tissue array construction
u. FREQUENTLY ASKED QUESTIONS
APPENDIX 1. Protocols for Pathology Reporting and Sample Collection
APPENDIX 2. Material Specification of the barcoded labels for glass slide
APPENDIX 3. Shipment Notification Procedure For Paraffin Embedded Tissue
APPENDIX 4. TMAs Supplies/Reagents Ordering Checklist
REFERENCE
Colorectal Cancer Family Registry - Coordinating Center Laboratory
University of Southern California, Department of Preventive Medicine
Drafted October 10, 1998
Revised Jan 29, 2004 AD
27
For USC CCFR PHASE III Study, due to the newly revised protocol for the adaptation of
TMA (tissue micro-array), the requested paraffin-embedded tissue (PET) will come only
as a block, (CCF will be the only exceptional site that blocks will not be available for
USC lab to process, they will continue to send the PET material in sections of the block).
 Information needed for selecting PETs:

For TMA:
If a PET block is available for the registry to retain, we will perform the TMA and keep the block in our
repository. The pathology lab from the site will select the tissue block as follows:
a. The preferred minimum dimension of the tissue is 4 mm x 4 mm x 4 mm.
b. Consist of approximately 70-80 % tumor cells.
c. Although normal DNA can be obtained from blood samples, blocks or sections should be selected to
have some normal cells. If available, a block of normal tissue should be retained for each patient,
particularly if deceased and/or if blood is not available.
For sites that a PET block is not available for the registry to retain and the pathology lab is unable to
perform the TMA, we request that a block be sent to us to process. Selection criteria of blocks for
sectioning are the same as the above.
The block will be returned within one month from the date that we received it.
Note: Fixative type has a direct bearing on the quality of extracted DNA.
i, Ethanol fixation: Although not routinely used in most pathology laboratories, it is
an especially good preservative of DNA.
ii, Mercury chloride: It contains fixatives such as B5 that have a deleterious effect
on DNA integrity. However, it is sometimes adequate for PCR.
iii, Bouin’ s solution: This also has a deleterious effect on DNA integrity. Just like
B5, it is sometimes adequate for PCR.
Duration of fixation is also important. Fortunately, PETs are routinely processed and fixed for less than
24 hours.
However, the inhibitory effects of certain fixatives can be overcome by further phenol-chloroform
extraction from DNA extracts.

CCF Only.
A PET block is not available for the registry to retain, we request:
a. 10 sections with a thickness of 5 microns for immunostaining.
b. 16 sections with a thickness of 5 microns for the microdissecting of DNA.
c. 4 sections with a thickness of 5 microns for the extraction of composite DNA.
d. 2 sections with a thickness of 5 microns for H&E (hematoxylin and eosin) staining.
Please refer to the following protocol for section preparation
The tests that will be performed on the PETs are destructive. Therefore, the slides or sections
will not be returned.
All remaining unused sections should be returned.
e. Selection criteria of blocks for sectioning are the same as the above.
Colorectal Cancer Family Registry - Coordinating Center Laboratory
University of Southern California, Department of Preventive Medicine
Drafted October 10, 1998
Revised Jan 29, 2004 AD
28
 Accessing the paraffin-embedded-tissue block/slides
a. Tracking
1. The lab MUST be notified prior to the shipment.
2. Include the following information in your e-mail.
 Send to all personnel below:
Ofeliya Avetisyan:
Qingru Feng:
Anh Diep:
E-mail: oavetisy@usc.edu; Tel #: (323) 865-0583
E-mail: qfeng@usc.edu;
Tel #: (323) 865-0581
E-mail: atdiep@usc.edu; Tel #: (323) 865-0713
 Attach a copy of the standard lab TUMOR SHIPPING LOG sheet.
 Provide the FedEx tracking #.
3. Package should be sent to the lab using the following shipping address:
Anh Diep c/o Haile’s Lab
USC Keck School of MED – Norris Cancer Center
Topping Tower 5421, 5427 and 5429
1441 Eastlake Ave.
Los Angeles, CA 90033
Lab Line #: (323) 865-0585
Fax #: (323) 865-0140
4. The lab will acknowledge the arrival of the package immediately upon receiving
(with a “cc” to Pat Harmon). Then follow with a report of the tumor accessing
status within the next few days.
5. After processing (one month turn around time), the lab will be responsible to
return the tissue to the coordinators.
6. As soon as the coordinators receive the package, the lab requests a confirmation sheet (the form
will be enclosed in the package) signed and faxed back to the lab for the return of the material.
b. Accessing
1. Have a complete pathology report with all pages.
2. Thoroughly deface all subject personal information: name, social security
number, and date of birth.
3. Pathology report should be readable.
4. Highlight the pathology # and the study ID #.
5. Log in the “LOG BOOK” the number of the items received.
6. Take note for any discrepancy.
7. Report to the center immediately the discrepancy, then follow up on all
correction actions.
8. Fill out ALL information (don’t leave blank) in a block processing log sheet
(including information of tissue source).
9. In case of a tissue that is not ON LOAN and we can keep the block, create a
separate logsheet for the block with a different Specimen ID.
10. Make a copy of the shipment log and staple them together with the block
processing log sheet for every single block.
Colorectal Cancer Family Registry - Coordinating Center Laboratory
University of Southern California, Department of Preventive Medicine
Drafted October 10, 1998
Revised Jan 29, 2004 AD
29
 Protocol for paraffin-embedded-tissue section preparation:
(For CCF Only)

Reagents and Equipment:
Alcohol, 75%--in a spray bottle
1.5 ml microcentrifuge tube
Permanent marker
Regular slide
ProbeOn Plus slide
Xylene
Microtome
Microtome blade
Floating bath
Gloves
Kimwipe
Toothpick
Tweezers
Brush
A. Order of the paraffin-embedded-tissue sections
Section #
Thickness in
microns
5
Uses
Prepared as
H & E stained
regular slide
2-11
(10 sections)
5
future immunohistochemical analyses
ProbeOn Plus slide
12-27
(16 sections)
5
DNA extraction
regular slide
28-37
(10 sections)
optional
5
DNA extraction
regular slide
38-41
(4 sections)
5
extraction of composite DNA
42
5
H & E stained
unmounted on any slides
each section is placed in
a 1.5 ml micro tube*
regular slide
1
*use regular slides to mount the section, if it is unfeasible to work with microtubes.
Colorectal Cancer Family Registry - Coordinating Center Laboratory
University of Southern California, Department of Preventive Medicine
Drafted October 10, 1998
Revised Jan 29, 2004 AD
30
B. Preparation of the paraffin-embedded-tissue sections
DNA-based experiments where PCR is widely used require cells free from attached molecules to be an
effective substrate for the molecular analysis. The purity of cells from paraffin-embedded tissue is
essentially equivalent to analyzing pure cell lines. This list of precautions must be followed to prevent
carryover from one specimen to another.
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
Slides and microcentrifuge tubes should be labeled with markers that stay permanent when in
contact with EtOH and xylene.
Label slides and tubes with the block id and section numbers according to the order of sectioning.
Use adhesive (ProbeOn) Plus slides or comparable slides for immunostaining.
Use regular slides for both H&E staining and microdissecting of DNA.
Air-dry all slides. DO NOT bake slides.
Use 1.5 ml microtubes for future constitutional DNA extraction. Use regular slides to mount the
section, if it is unfeasible for the pathology lab to work with micro-centrifuge tubes.
Only touch the edge of slides.
Completely place the PET sections within 1.5 ml microtube without overhang.
Do not touch the rims or insides of caps when opening and closing tubes.
The microtome and work area are cleaned twice with ethanol or xylene between specimens to
remove excess paraffin, tissue fragments and debris.
The microtome blade is changed after sectioning each case.
Each block is refaced before sectioning.
Floating bath must be changed for each case.
The temperature for the bath: 48C to 52C.
Soaking the blocks on ice or a ice tray is required.
A new pair of disposable gloves will be used for handling and sectioning each case.
Brushes for picking up the sections must be cleaned with ethanol and UV radiated between each
case. Toothpicks may be used in place of brushes.
Drafted October 10, 1998;
Revised Feb 11, 2004 AD
Colorectal Cancer Family Registry - Coordinating Center Laboratory
University of Southern California, Department of Preventive Medicine
Drafted October 10, 1998
Revised Jan 29, 2004 AD
31
 The Alternative Protocol for Centers that cannot provide the
complete protocol with 42 sections:

Reagents and Equipment:
Alcohol, 75%--in a spray bottle
1.5 ml microcentrifuge tube
Permanent marker
Regular slide
ProbeOn Plus slide
Xylene
Microtome
Microtome blade
Floating bath
Gloves
Kimwipe
Toothpick
Tweezers
Brush
A. Order of the paraffin-embedded-tissue sections
Section #
Thickness in
microns
5
Uses
Type of Slides Used
H & E stained
regular slide
2-17
(16 sections)
5
DNA extraction
regular slide
18-27
(10 sections)
5
future immunohistochemical analyses
charged (ProbeOn Plus is
preferred) slide
28
5
H & E stained
regular slide
1
Drafted October 10, 1998;
Revised May 11, 2001 AD
Colorectal Cancer Family Registry - Coordinating Center Laboratory
University of Southern California, Department of Preventive Medicine
Drafted October 10, 1998
Revised Jan 29, 2004 AD
32
B. Preparation of the paraffin-embedded-tissue sections
DNA-based experiments where PCR is widely used require cells free from attached molecules to be an
effective substrate for the molecular analysis. The purity of cells from paraffin-embedded tissue is
essentially equivalent to analyzing pure cell lines. This list of precautions must be followed to prevent
carryover from one specimen to another.
1.
2.
Slides and microcentrifuge tubes should be labeled with markers that stay permanent when in
contact with EtOH and xylene.
Label slides and tubes with the block id and section numbers according to the order of sectioning.
3. Air-dry all slides. DO NOT bake slides.
4.
5.
Only touch the edge of slides.
The microtome and work area are cleaned twice with ethanol or xylene between specimens to
remove excess paraffin, tissue fragments and debris.
6. The microtome blade is changed after sectioning each case.
7. Each block is refaced before sectioning.
8. Floating bath must be changed for each case.
9. The temperature for the bath: 48C to 52C.
10. Soaking the blocks on ice or a ice tray is required.
11. A new pair of disposable gloves will be used for handling and sectioning each case.
12. Brushes for picking up the sections must be cleaned with ethanol and UV radiated between each
case. Toothpicks may be used in place of brushes.
Colorectal Cancer Family Registry - Coordinating Center Laboratory
University of Southern California, Department of Preventive Medicine
Drafted October 10, 1998
Revised Jan 29, 2004 AD
33
 Tissue Micro-array standard operating procedure(USC Phase III)
a. Equipment & Accessories
1. Manual Tissue Arrayer MTA-2 from Beecher Instruments, Inc. Sun Prairie, WI,
(To order: Tel # (608) 837-8847 or Fax # (608) 837-7118)
www.beecherinstruments.com;
support@beecherinstruments.com
Model MTA-2 includes:
 2 sets of 0.6mm punches
 1 recipient block holder, 11 additional will be ordered
 1 donor block bridge
 operating manual and tools
 two sets of punches: 0.6 and 1.0 mm; 1.5 and 2.0 mm.
 Optional: Depth Stop Kit (DSK)
2. A magnifying glass model Wave + A/R from LUXO, Port Chester, NY.
(Purchase can be processed through VWR); www.luxo.com
3. Tape sectioning system (Cat # PSA) from Instrumedics, Inc. Hackensack, NJ
(To order: Tel # (201) 343-1313); www.instrumedics.com
System includes:
 UV Curing Lamp, Cat # PSA-LMP
 PSA Curing Lamp bulb, Cat # PSA-BLB
 Hand roller, Cat # HR
 Voltage Transformer, Cat # PSA-TR
 TPC Solvent can, Cat # PSA-CAN
 CureMount II, Cat # CMII (60 ml/bottle)
b. Reagents and supplies
Donor blocks
Glass slides
Blank (recipient, array) block
Regular paraffin
Mold (for preparation of blank block), 5-10mm thick.
Cassette (as above)
Plastic block holder
Knife
Alcohol, 70% (in a small bottle)
Kimwipes, small
Colorectal Cancer Family Registry - Coordinating Center Laboratory
University of Southern California, Department of Preventive Medicine
Drafted October 10, 1998
Revised Jan 29, 2004 AD
34
THIS IS THE EQUIPMENT WE HAVE
MANUAL ARRAYER (MTA-II)
Colorectal Cancer Family Registry - Coordinating Center Laboratory
University of Southern California, Department of Preventive Medicine
Drafted October 10, 1998
Revised Jan 29, 2004 AD
35
c. Preparation of the blank recipient array block
1. Melt regular paraffin at temperature 55-580C, pour the melted paraffin into a mold.
3. Place a cassette on top of the melted paraffin until the complex is cooled. The
mold is ready to be removed.
4. Cut all irregularities from the plastic block holders and check for any holes (air bubbles) in the block.
d. Preparation of the donor blocks and slides
1. Prepare a fresh H&E– stained slide from each block.
2. Circle or mark the sampling sites on the corresponding H&E slide.
e. Designing and constructing the Array (with cell carry-over free instruction).
1. Each recipient block will contain 384 samples divided into four quadrants.
2. 1.0 mm spacing between the centers of two adjacent samples is maintained. This
will make
adding increments to the micrometer a lot easier.
3. Leave 2.5mm margins on the top and the bottom of the array block.
4. Leave 3.0mm on the left and the right edge of the array block. (See worksheet “Diagram of TMA
organization: Design of the array with positions of the punches)
5. Insert the empty recipient block in the holder and tighten the clamping screws to
prevent the block from slipping.
6. Place the holder firmly against the location curbs.
7. Make the hole in the first position in recipient block (at this point, the
micrometer should be set to zero). *The smaller punch is used to create
the hole.
8.Align the H&E slide and the corresponding tissue block. Retrieving the sample from the donor and
empty the tissue core into the hole created by the smaller punch. *The larger punch is used to take
sample tissue.
9. Prepare the array (recipient) blocks according to the preplan TMAs organization.
10. Between cases, to ensure cell carry-over free for subsequent DNA_PCR-based
block to clean up the larger punch.
analysis, punch at least 10 times into blank
11. Make sure the punched-blank-paraffin core including its residual cleanly empty from each punch. To obtain the best
cleaning result, creating a small horizontal motion of the punch by slightly moving the turret to help break the punch free
from the inserted tissue core.
f. Align the punch
1. Check the positioning and alignment of punches before beginning to construct the first array.
2. To verify alignment, place a blank paraffin block in the block holder and put this complex in position
on the arrayer as if it were a recipient block.
3. Press the smaller punch down until it leaves a mark in the paraffin surface.
4. Move the turret to switch the larger sampling punch in position, and again make a mark on the
paraffin surface.
5. Adjustment is needed if the marks are not coincidental.
6. Front to back alignment is adjusted by setscrews recessed in the inboard side of the plastic v-blocks
holding the punches.
7. To correct left-right alignment, use the largest hex key to turn the left-right setscrews on each lower
side of the turret.
Colorectal Cancer Family Registry - Coordinating Center Laboratory
University of Southern California, Department of Preventive Medicine
Drafted October 10, 1998
Revised Jan 29, 2004 AD
36
g. Complete PET processing protocol and the order of the PET banking procedure
PET
Banking
task order
1st
# of arrays
or
Section #
1 section
donor block
Thickness in
microns/diameter
in mm
5 um
Uses
Prepared as/ for
H & E stained
regular slide
blocks and areas selection
2nd
121.5 ml micro
tubes
0.6 mm cores
8T + 4 N cores per
donor block
Direct extraction
of composite DNA
0.6 mm - 3 cores
per donor block
1 adenomas, 1 tumor and 1 normal
25% duplicates from 48 tissue of larger
tumors
oriented with a set of 3 normal cores in
quadrant A of the block:
central area of selected region
NOT inserted in TMAs
each core is placed in
a 1.5 ml micro tube
1 A/C/N TMA
(Adenomas, Carcinomas and
normal array)
A set of 3 A/C/N TMAs will
be made if adenomas tissue is
available, always include 25%
of duplicates
3rd
1 array block
Array ID
labeled
ACN01
A1a, A1b, A1c
4th
4 array blocks
0.6 mm-4 cores per
donor block
Array ID
subdivision
labeled A to H
1 tumor and 1 normal,
190 pairs
25% duplicates from 24 tissue of larger
tumors
oriented with 4 blank spots in 4 quadrants,
localization:
A1a, B1a, C1a and D1a
5th
8 array blocks
Array ID
subdivision
labeled I to P
0.6 mm-4 to 8
cores per donor
block
1 tumor core per array block
96 duplicate pairs adjacent to each other
(randomly localized) as a QC
25% duplicates from 48 tissue of larger
tumors
oriented with 3 normal cores in 3 quadrants,
localization:
4 T/N TMAs
(Tumor and Normal arrays)
totally 166 subjects with 24
subjects in duplicate
A set of 2 T/N TMAs,
make 4 sets
4 to 8 T-only TMAs
(Tumor only arrays)
totally 332 subjects per block
with 48 subjects in duplicate
A1a, B1a, C1a
1 breast tissue core in D1a
6th
7th
8th
*9th
1 section
donor block
5 sections
donor block
10 sections
donor block
5 um
H & E stained
5 um
DNA analyses
5 um
IHC analyses
1 section
per array
recipient block
5 um
H & E stained
Taping-sectioning method
regular slide
punched spots verification
regular slides
for QC
ProbeOn Plus slides
for QC
Finished full TMAs (332)
4 T/N TMAs, 8 T-only TMAs
and 1 A/C/N TMA
13 Adhesive coated slides
for spots confirmation and
localization
*cell carry-over free procedure conducted between cases.
Colorectal Cancer Family Registry - Coordinating Center Laboratory
University of Southern California, Department of Preventive Medicine
Drafted October 10, 1998
Revised Jan 29, 2004 AD
37
h. After the construction of a full array block is completed and before sectioning, the array block surface
must be smooth and leveled.
1. Place the finish recipient array block in a warm chamber (37C) for 10-15 min to promote
adherence of the tissue to the walls of the holes in the array block and make wax flexible to handle.
2. level the surface of the block after it has warmed by a clean glass slide. Apply even pressure to
push all tissue cores on the array to the same level.
3. The array block is now ready for sectioning.
i. Sectioning the blocks (donor and array).
1.
2.
3.
4.
Use standard microtome sectioning techniques to cut the sections of the donor block.
Use a adhesive-coated tape sectioning aid system to collect the H&E stained section from each
recipient block.
Use current routing technique to collect sequential sections from the punched donor block: the first
after-punched section is for H&E staining, the rest of the sections will be mounted on uncharged
slides.
Use the tape-sectioning aid to cut the H&E section for the array recipient blocks.
j. Tape-sectioning the array block 0.6 mm in diameter and spacing between two adjacent array
Paraffin tape-transfer slides
Tape windows
TPC solvent
CureMount II
Ultra-violet curing lamp
PSA curing lamp bulb
Hand roller
Voltage transformer
TPC solvent can
1. Trim the paraffin block to expose the tissue face.
2. Remove the section of mylar that says “ PEEL THIS OFF” from the pink tape to expose the
adhesive layer. Adhere the tape window to the block face, using the handle roller with firm
pressure. Be sure not to trap bubbles. If bubbles are trapped remove the tape and try again.
3. Using a sharp blade cut the section, which is supported and captured by the tape.
4. Laminate the tape with the section-side-down onto the adhesive-coated slide using the hand roller.
Be sure that you have removed the mylar film from the slide. The lamination step is very
important. Be sure the section is flat against the adhesive layer and that bubbles are not trapped
under the section. Check for bubbles by examining the underside of the slide.
5. Place the slide, section-side-down, on the Curing lamp tray. 15 slides fit comfortably on the tray.
6. Turn on the UV light and the tray under the lamp. Polymerization of the adhesive coating into a
plastic layer usually takes 30-60 seconds. If the coating is not polymerized, leave slied under the
UV light for a longer time.
7. Immerse the slide, with the tape window still in place, in the TPC solvent in the solvent can.
IMMEDIATELY REVOVE THE TAPE, by slowly and carefully peeling it away from the slide.
Solvent should be between the slide and the tape as you peel the tape away.
8. The slide is ready to be deparaffinized. Some paraffin need a longer time than others in xylene to
completely deparaffinize. Once hydrated the section is ready for your protocol. For H&E staining
see the enclosed instructions.
(When ready to cover slip dehydrated and cleared sections, use the CureMount mounting medium
included with system. Instructions are enclosed.)
k. Hematoxylin And Eosin Staining Procedure: Hematoxylin and eosin staining is performed on a regular
basis on all received specimens as a first step in the diagnostic procedure. Specimen type:
1. Frozen tissue may or may not be embedded in O.C.T.
2. Paraffin blocks or precut slides obtained from referring laboratories.
Colorectal Cancer Family Registry - Coordinating Center Laboratory
University of Southern California, Department of Preventive Medicine
Drafted October 10, 1998
Revised Jan 29, 2004 AD
38
Reagents:
3.
4.
5.
6.
7.
8.
Acetone. ( See Standard Operating Procedure for Hazardous Chemicals, LAB SAFETY SECTION
). Store at room temperature.
Harris Hematoxylin Solution Modified ( Sigma Diagnostics Cat. #HHS-32). Store at room
temperature until expiration date.
1% Acid Alcohol ( 100 parts 70% ethanol + 1 part 37% hydrochloric acid ). Store at room
temperature for 12 months.
Scott’s Tap Water Substitute Concentrate “10x” ( 1 part Scott’s Tap Water + 9 parts distilled
water). ( Sigma Diagnostics Cat. # S 5134). Store at room temperature until expiration date.
Eosin Y Solution, Alcoholic ( Sigma Diagnostics Cat # HT 110-1-32). Store at room temperature
until expiration date.
95%, 100% Ethanol. Store at room temperature.
Stepwise Procedure:
9.
Paraffin slides are dried in a 60- 65C over for at least an hour. Deparaffinize sections in FRESH
xylene (35 min); rehydrate in graded ethanol (2 min each) and transfer to distilled water. (Frozen
sections are fixed in picric acid para-formaldehyde or in 95% alcohol for 15 minutes, then
transferred to distilled water.)
Stain the slides in hematoxylin solution for 5 minutes.
Rinse in running tap water, to remove excess hematoxylin, for 2 minutes.
Differentiate in acid alcohol for 15 seconds.
Rinse in running tap water for 2 minutes.
Put the slides in Scott’s tap water substitute for 30 seconds – 1 minute.
Rinse in running tap water for 2 minute.
Counter stain the slides in eosin for 3 minutes.
Rinse in 2 changes of 95% ethanol, 2 changes 100% ethanol ( 2 minutes ).
Rinse in 3 changes of xylene ( 2 minutes each).
Cover slip with permount.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
Result:
 Nuclei will stain “Blue”
 Cytoplasma will stain into various shades of “pink”, identifying different tissue components.
References for the H&E staining procedure:
1.
Luna LG(ed.). Routine Staining Procedures: Hematoxylin and Eosin Stains in Manual of
Histologic Staining Methods of the Armed Forces Institute of Pathology. Third Edition. McGrawHill book Company. 1968; 32-41
2. Sheehan DC, Hrapchak BB. Theory and practice of Histotechnology. Second Edition. Battelle
Press. Columbus, Richalnd. 1987; 144-5.
Troubleshooting for the H&E staining procedure:
 If sections appear too blue at step 7, re-immerse them in acid alcohol for a few seconds, then
repeat steps 5 to 7 and recheck.
 If sections appear too plate at step 7, re-immerse them in the hematoxylin for further staining, then
repeat step 3 to 7.
 The alcohol used in step 9 serve a dual purpose: dehydration of the tissue, as well as removal of
excess eosin. By passing too rapidly through these alcohols, excess eosin will remain in the section
and overshadow many diagnostic features.
 Occasionally, the used reagents are know to be working satisfactorily, yet some sections fail to
show nuclear staining. This can be due to:
a) autolysis of the tissue section,
b) prolonged storage of the wet tissue in non-buffered formalin,
c) dried or burned tissue.
Basophilic staining properties may be restored by treatment of the hydrated sections with
one of the following solutions:
(1). 5% aqueous sodium bicarbonate overnight followed by a 5- minutes wash in tap
water before staining, or
Colorectal Cancer Family Registry - Coordinating Center Laboratory
University of Southern California, Department of Preventive Medicine
Drafted October 10, 1998
Revised Jan 29, 2004 AD
39
(2). 5% aqueous periodic acid overnight, followed by 3 changes of distilled water before
staining.
l. Instruction to use the four block indexer (OPTIONAL for MTA-I only)
The Four-Block Indexer (4BI), which holds four standard recipient blocks in its turntable
simultaneously, make it easier for users to construct concurrent arrays. With the 4BI in place, each of the
four different recipient blocks may be quickly indexed into position.
Introduction
1. Insert the 4BI in position, it has three slots on the underside. Any one of these slots fits over the
shorter silver bar that braces the standard recipient block holders (RBH) in place.
2. Circular finger rests for rotating indexer, thumb rest corner for swinging bridge plate to the left,
access grooves for setscrews.
3. The cassettes of array blocks are held in the pockets of the turntable with a pair of setscrews for
each block.
m. Instruction to use the depth stop kit
The Depth Stop Kit (DSK) provides the additional capabilities of: (a) controlling the depth of the donor
punch in the donor blocks, and (b) holding the donor punch just above the surface of the recipient block
during the transfer of the tissue core from the donor punch to the recipient block.
Introduction
1. The DSK includes 5 aluminum (silver color) donor transfer stops and 5 brass (yellow color) donor
block stops. Each is numbered with the depth in millimeters, 2,3,4,5, or 6. If wishing to extract 3
mm long cores and place them in 3 mm deep holes in the recipient block, and then choose the #3
donor transfer stop and the #3 donor block stop.
2. The donor transfer stop fits on top of the depth stop block, with the higher end toward the back.
3. The donor block stop fits directly onto the donor punch hub, with its number upright and toward
the user. The circular part of the donor block stop placed underneath the punch hub so that the
punch needle goes through the slot in circular part.
4. To operate the donor block stop, simply move the turret downward in the usual manner, until the
bottom of the donor block stop contacts the top of the donor block. The needle will now be the
selected depth into the donor block.
n. Maintenance of the Tissue Arrayer
1. Use Kimwipe to clean any residual paraffin from the punches, sampling block and
block
holders.
2. Use alcohol (or xylene ) to wipe parts. DO NOT SOAK ANY PARTS, PUNCHES IN A
SOLVENT. DO NOT USE SUCH A SOLVENT FOR CLEANING THE X-Y OR Z RAILS.
3. Punches need to be periodically replaced after several hundred or a thousand punches.
o. ID assignment system
1. Specimen ID is given sequentially by the database. This ID will be labeled on the donor blocks and
to the slides. The ID numbering system consists of a first 3 digits “121”, which is coded for
“BLOCK”, while “120” is coded for “BLOOD”; with a hyphen to separate the subsequent 6
numerical digits, to uniquely identify each individual block. To the slides, there will be an
additional sub # to identify the order of the section. Therefore, the Specimen ID will look like:
121-123456-01
2. Array Specimen ID is given by the TMA operator using a system of 4 numerical digits and 1
alphabetical digit. The first 4 numerical digits will be coded for an array set of replicated blocks
derived from a set number of donor blocks (332 blocks totally). The 5 th alphabetical digit is used to
identify the type of arrays, either they are consisted of tumor/normal tissues (T/N) or tumor tissue
only (T-only) or adenomas/carcinomas/normal tissues (A/C/N). A to H have been used to assign
for 8 of the replicate T/N array blocks and I to P have been used to assign for 8 of the replicate Tonly array blocks. Except for A/C/N array blocks, the ID system will be different. Each A/C/N
array block will be labeled as ACN01 sequentially as it will be made for blocks that adenomas
tissues are available. Therefore, the Array Specimen ID will look like:
Colorectal Cancer Family Registry - Coordinating Center Laboratory
University of Southern California, Department of Preventive Medicine
Drafted October 10, 1998
Revised Jan 29, 2004 AD
40
1234A to 1234H for T/N array blocks
1234I to 1234P for T-only array blocks
ACN1 for A/C/N array blocks
p. Worksheets
3. PET block accessing log. Worksheet 1
4. Localization checkerboard of the T/N TMAs. Worksheet 2
5. Localization checkerboard of the T-only TMAs. Worksheet 3
4. Localization checkerboard of the A/C/N TMAs. Worksheet 4
 Notes: If a hole in one array block was damaged, skip that hole which means one replicate
block lesser for the set of array blocks. If the tumor is large enough to make an extra series,
these duplicates will consider the QC for the assay. In any mean, always make the same
localization in a set of array blocks for the same donor tumor.
5. Diagram of the TMA organization. General
6. Design of the array: position of punches
7. Diagram of the TMA organization. Example of localization assignment
8. Diagram of the TMA organization. T/N array
9. Diagram of the TMA organization. T-only array
10. Diagram of the TMA organization. A/C/N array
11. PET blocks and slides log sheet. Worksheet 5
Colorectal Cancer Family Registry - Coordinating Center Laboratory
University of Southern California, Department of Preventive Medicine
Drafted October 10, 1998
Revised Jan 29, 2004 AD
41
Worksheet 1. PET block accessing log.
PET BLOCK ACCESSING LOG
Date
Received
Center
Block ID #
# of Blocks
Received
Date/Tech
Screened
Date/Tech
Processed
Date/Tech
Returned
Comment
Worksheet 2. Localization checkerboard of the T/N TMA (X for spotted, 4 duplicates/ quadrant)
Colorectal Cancer Family Registry - Coordinating Center Laboratory
University of Southern California, Department of Preventive Medicine
Drafted October 10, 1998
Revised Jan 29, 2004 AD
42
Array
Specimen ID
1
2
3
4
QUADRANT
1
(circle):
1 1 1
A
1
1
B
2
1
1
1
3
1
C
1
1
4
1
1
1
5
D
1
1
1
6
1
1
1
7
1
1
1
8
a
b
c
d
e
f
g
h
i
j
k
l
# OF DONOR BL _______________________________________________________________________________________________________________________________________________
# OF DUPLICATE______________________________________________________________________________________________________________________________________________
Colorectal Cancer Family Registry - Coordinating Center Laboratory
University of Southern California, Department of Preventive Medicine
Drafted October 10, 1998
Revised Jan 29, 2004 AD
43
1
Worksheet 3. Localization checkerboard of the T-only TMA (X for spotted, 8 duplicates/quadrant)
Array
Specimen ID:
1
5
QUADRANT (circle):
8 8 1 8 8 8 2
2
6
3
7
A
8
8
8
B
3
8
8
8
4
8
C
4
8
8
8
D
5
8
8
8
6
8
8
8
7
8
8
8
8
a
b
c
d
e
f
g
h
i
j
k
l
# OF DONOR BL _______________________________________________________________________________________________________________________________________________
# OF DUPLICATE______________________________________________________________________________________________________________________________________________
Colorectal Cancer Family Registry - Coordinating Center Laboratory
University of Southern California, Department of Preventive Medicine
Drafted October 10, 1998
Revised Jan 29, 2004 AD
44
8
Worksheet 4. Localization checkerboard of the A/C/N TMA (X for spotted)
Array
Specimen ID
QUADRANT (circle):
1
1
1 1
1
1
2
A
1
B
1
1
3
1
C
1
1
4
1
D
1
1
5
1
1
1
6
1
1
1
7
1
1
1
8
a
b
c
d
e
f
g
h
i
j
k
l
# OF DONOR BL _______________________________________________________________________________________________________________________________________________
# OF DUPLICATE______________________________________________________________________________________________________________________________________________
Colorectal Cancer Family Registry - Coordinating Center Laboratory
University of Southern California, Department of Preventive Medicine
Drafted October 10, 1998
Revised Feb 13, 2004 AD
1
Diagram of TMA organization. General
a
b
c
d
e
f
g
h
i
j
k
l
B
a
b
c
d
e
f
g
h
i
j
k
l
8
8
7
7
6
6
5
5
4
4
3
3
2
2
1
A
a
b
c
d
e
f
g
h
i
j
k
l
1
8
8
7
7
6
6
5
5
4
4
3
3
2
2
1
1
a
b
c
d
e
USC Genetic Epidemiology Laboratory
Keck School of medicine, department of Preventive Medicine
Colorectal Cancer Family Registry - Coordinating Center Laboratory
University of Southern California, Department of Preventive Medicine
D
f
g
h
i
j
k
l
Date drafted: April 21, 2004
Anh T. Diep
Drafted October 10, 1998
Revised Feb 13, 2004 AD
C
8.8
8.8
Design of the Array : Position of the punches (size in mm)
0.8
5.2
a
b
c
d
e
f
g
h
i
j
5.2
5.2
l
a
k
8
5
5.2
b
c
d
e
f
g
h
i
j
k
l
8
Y=22
11
7
0.8
0.2
7
6
6
5
5
4
4
3
3
5.6
B
2
1
1
D
2
1
0.8
1
R = 0.3
1
11
1
X=35
8
A
5
a
b
c
d
e
f
g
h
4
i
j
k
l
7
7
6
6
5
5
4
4
3
3
2
2
1
1
0.8
4
8
0.8
a
b
c
d
e
f
h
4
i
j
k
l
4
8.8
Colorectal Cancer Family Registry - Coordinating Center Laboratory
University of Southern California, Department of Preventive Medicine
g
8.8
Drafted October 10, 1998
Revised Feb 13, 2004 AD
11
C
5.6
11
Diagram of TMA organization. Example of Localization assignment.
a
b
c
d
e
f
g
h
i
j
k
l
B
a
b
c
d
e
f
g
h
i
j
k
l
8
8
7
7
6
6
5
5
4
4
3
3
2
D
2
1
1
Localization
A7g
A
a
b
c
d
e
f
g
h
i
j
k
l
8
8
7
7
6
6
5
5
4
4
3
3
2
2
1
1
a
b
c
d
e
USC Genetic Epidemiology Laboratory
Keck School of medicine, department of Preventive Medicine
Colorectal Cancer Family Registry - Coordinating Center Laboratory
University of Southern California, Department of Preventive Medicine
f
g
h
i
j
k
l
Date drafted: April 21, 2004
Anh T. Diep
Drafted October 10, 1998
Revised Feb 13, 2004 AD
C
j
k
l
k
l
i
i
j
h
f
f
h
e
e
g
d
d
g
c
c
4
5
6
7
8
b
l
l
b
k
k
a
j
j
7
6
5
4
3
3
blank
4
5
6
7
8
blank
blank
1
2
2
1
blank
8
7
6
5
4
3
3
USC Genetic Epidemiology Laboratory
Keck School of medicine, department of Preventive Medicine
Colorectal Cancer Family Registry - Coordinating Center Laboratory
University of Southern California, Department of Preventive Medicine
Drafted October 10, 1998
Revised Feb 13, 2004 AD
blank
C
blank
blank
A
a
1
2
2
1
blank
a
i
i
f
f
h
e
e
h
d
d
g
c
c
g
b
b
8
a
B
D
Diagram of TMA Organization. Tumor-and-Normal TMA Location
Date drafted: April 22, 2004
Anh T. Diep
i
j
k
l
i
j
k
l
f
f
h
e
e
h
d
d
g
c
c
g
b
b
l
l
a
k
k
7
6
7
4
5
6
5
3
4
2
3
1
2
1
8
Breast
Normal
8
2
3
4
5
6
7
7
6
5
4
3
2
USC Genetic Epidemiology Laboratory
Keck School of medicine, department of Preventive Medicine
Colorectal Cancer Family Registry - Coordinating Center Laboratory
University of Southern California, Department of Preventive Medicine
C
Normal
A
1
1
Normal
a
j
j
8
8
i
f
f
i
e
e
h
d
d
h
c
c
g
b
b
g
a
a
B
D
Diagram of TMA Organization. Tumor-only TMA Location
Drafted October 10, 1998
Revised Feb 13, 2004 AD
Date drafted: April 22, 2004
Anh T. Diep
j
k
l
j
k
l
i
i
f
f
h
e
e
h
d
d
g
c
c
g
b
l
l
b
k
k
7
6
2
3
4
5
6
5
4
3
2
8
1
1
8
C
A
a
1
2
3
4
5
6
7
7
6
5
4
3
2
1
Normal
a
j
j
8
i
f
f
i
e
e
h
d
d
h
c
c
g
b
b
g
a
7
8
a
B
D
Diagram of TMA organization. Adenomas-Carcinomas-and-Normal TMA Location
USC Genetic Epidemiology Laboratory
Keck School of medicine, department of Preventive Medicine
Colorectal Cancer Family Registry - Coordinating Center Laboratory
University of Southern California, Department of Preventive Medicine
Drafted October 10, 1998
Revised Feb 13, 2004 AD
Date drafted: April 22, 2004
Anh T. Diep
PET slides log sheet. Worksheet 5 (Available only from the ACESS database)
Colorectal Cancer Family Registry - Coordinating Center Laboratory
University of Southern California, Department of Preventive Medicine
Drafted October 10, 1998
Revised Feb 13, 2004 AD
q. Barcode labels (NOT ADAPT YET)
1. For the array recipient blocks: (Making 2 labels per array block/ Array Spec ID)
 L-1002-10 laser printable sidewall Tough-tags 0.25” x 1.5” (prefer to be smaller 0.25” x 1.0” and clear instead
of white) will be used.
 This specially designed labels is super durable. They are carefully engineered to strongly adhere to all plastics
and other materials. Labels will withstand organic solvents, caustic agents, humid incubators, boiling water
baths, refrigerators and freezing temperatures without peeling.
 Our lab has routinely use them for PCR plates which have undergone cycles of different temperatures and then
long term stored in –20°C or -80°C.
 This label is now applied on the wax cassette that hold PET tissue. The cassettes will be undergone icy cold
water soaking before sectioning and then will long term stored at controlled ambient atmosphere or 4°C
condition.
 Therefore, a simple test has be performed in our lab by soaking the tough-tag labeled paraffin cassette in cold
water for many hours, then observe the adhesive by peeling and the writing (laser printing) for it legibility. The
result has confided our choice for this purpose.
2. For the glass slides: (Randomly, uniquely for each slide, hand label the slide sequential #)
 A pre-printed barcode FLAP labels will be applied to the glass slides of this study.
 This FLAP Label was designed for use on microscope slides.
 The FLAP Label construction was developed for use in the laboratory and other high stress, chemically rigorous
environments, such as cytology, histology and cytogenetics.
 Material Specification TS822 (Appendix 3) describes the list of chemicals and stains that these labels have been
tested for.
 The feature of FLAP uniquely designed with an overlaminate flap that is placed over the image after printing for
additional image protection against harsh chemical environments.
 The adhesive is formulated to permanently bond to most demanding surfaces and microscope slide will be one
of them (label should be applied to the painted or frosted sections of glass slides to insure maximum bond.
 To assure the described performance, our lab tested the labels on 10 tested microscopic glasses using a simple
design: After applying the labels on the slides, barcode scanned the specimen ID, then conducting our routine
H&E staining procedure in which the following chemical and temperature have been included:
In 60- 65C for at least an hour >> In FRESH xylene 15 min >> Rehydrate in graded ethanol ~10 min >>
Transfer to distilled water >> Stain the slides in hematoxylin solution for 5 minutes >> Rinse in running tap
water for 2 minutes >> Differentiate in acid alcohol for 15 seconds >> Rinse in running tap water for 2 minutes
>> Put the slides in Scott’s tap water substitute for 30 seconds – 1 minute >> Rinse in running tap water again
for 2 minute >> Counter stain the slides in eosin for 3 minutes >> Rinse in 2 changes of 95% ethanol for 2
minutes each >> Rinse in 2 changes 100% ethanol for 2 minutes each >> Rinse in 3 changes of xylene for 2
minutes each >> After above conditions, the slides were scanned again to examine the quality of printing.
 The adhesive was also carefully examined by physical peeling. The test confide our choice of labels for this
study.
 Preprinted information is designed specifically to have 11 digits in total with the last digit a "MOD 10-human
readable interpretation" check digit. There is also a capital letter that shows in front of barcode number. This
series of a letter followed with 10 sequential digits plus a MOD-10 check digit are all human-readable on the
label that is scanned. The printing symbology code is 128.
 The product cat. #: TS822. Provided by: DELTA-ONE Software, Inc. (2841-G Saturn St. Brea, CA 92821,
Telephone # (714) 528-7226, Fax # (714) 528-7236, Sale Rep. For USC-CFR is Marilyn Fox)
 The graphic design of the label approved by our lab is (this is only a sample label temporarily used for the first
10,000 cryovials, the final approved label is described above):
Colorectal Cancer Family Registry - Coordinating Center Laboratory
University of Southern California, Department of Preventive Medicine
Drafted October 10, 1998
Revised Feb 13, 2004 AD
7/8”
USC Genetic Epi
3/4”
A1234567890
3.
Other labels need for worksheet, donor blocks, etc will use AVERY common office type labels.
r. Storage
1. store the array recipient blocks in a embedding ring filing cabinet brand name Sakura Finetek Tissue-Tek. This stackable
cabinets accommodate 1000 rings/blocks. File the blocks chronologically. Store at room temperature in a climate
controlled laboratory or 4C walk in cold box. (purchase information: VWR cat # 25608-866)
2. store the slides in a steel micro slide storage cabinet “Boekel”. It is also a stackable unit with interlocking tabs and
removable drawers for transporting to work areas. The 6-drawer cabinet can hold up to a total of 5000 standard micro
slides. The slides are filed and stored at room temperature in a climate controlled laboratory. (purchase information:
VWR cat # 48466-500)
s. Data File (TO COME)
t. Software for Spot Imaging Documentation (TO COME)
Colorectal Cancer Family Registry - Coordinating Center Laboratory
University of Southern California, Department of Preventive Medicine
Drafted October 10, 1998
Revised Feb 13, 2004 AD
u. TROUBLESHOOTING guide for manual tissue array construction
1. When depositing the samples to the recipient (tissue array) block, the tissue core
does not seem to go exactly to the location of the hole created for it with the small
punch.
 Make sure that you are not accidentally moving the turret while pushing down the sample.
 Punch alignment may have changed (for example, after replacing the punch). Realign the punch.
2. Spacing between adjacent array samples seems to vary, causing a distortion in the tissue array.
 Make sure the punch handles are always turned to a consistent position.
 The punch may be bent. Check it and change if necessary.
3. The punch does not extract a tissue or wax core sample.
 This situation can be corrected by gently pushing the stylet down against the sample before trying
to extract it (tamping). Placing pressure on the top of the sample causes it to compress axially and
expand radially, thus creating a tighter fit within the punch tube. Rotating the needle handle
slightly also helps break the sample from its base. Longer core samples are also easier to extract
than shorter core samples. This is particularly important with the larger punches (1.0, 1.5, and 2.0
mm diameter).
4. The tissue core does not come out of the punch easily. There seems to be resistance at the tip of the
punch.
 The sharpened edge of the punch tip is probably distorted. Change the punch.
5. The tissue core was accidentally pushed too deep and will cause a hole in the tissue array. Can
something be done to fix this?
 Yes. Just remove that sample with the small punch and place a new sample in the same position.
6. I have now arrayed 200 specimens, and it seems like the middle part of the tissue array is higher than
the edges. It looks like a small hill. What causes this and can it be fixed?
 If the array block is made in very high density, there may be an accumulation of residual paraffin,
partly caused by the slight size difference between the sample tissue cores and the holes in the
array. This makes the middle part of the tissue array protrude upwards, and this phenomenon
becomes visible after arraying a large number of samples. In essence, you may be trying to fit too
many samples in too little a space. To prevent this, either increase the spacing between adjacent
array samples or make the holes in the array block deeper. To flatten any existing outgrowths in
the tissue array, warm the block to 35-37°C for 15 minutes. While the block is still warm and
elastic, place a clean microscope slide on top of the array block, and carefully press downward
until all the specimens are at an even level. Let the block cool before removing the slide.
7. One of the micrometers is difficult to move

Make sure that both its locknuts are completely loose: the black knurled ring near the base of the
handle and the silver knurled knob at the side of the slide.

Tighten the turret pivot screw in 1/8-inch increments until the swinging turret motion is just
blocked--do not overtighten.

If the digital readout does not respond to the control buttons or knob, or if the readout is dim, the
micrometer battery may be too old and needs to be replaced. Batteries are located on the underside
of the micrometers, and are accessible through the circular holes in the base plate. Reposition the
arrayer carefully to gain access to the battery covers. Insert a new battery with the positive (+) side
away from the cover, tighten the cover, and check to verify that the micrometer numbers change as
you twist the handle. You may need to reinsert the battery two or three times before the battery
correctly activates the readout. We recommend Renata, Sanyo, or Duracell type 2450 batteries
because some other makes may not fit. A small booklet specifically about the micrometers is
included with the arrayer and will provide more information about the micrometers.

Recipient block holders are held very firmly in place by two magnets located on the base plate.
Two strips of tape also help hold the recipient block holders securely by increasing friction. Users
who exchange recipient block holders frequently may want to facilitate the exchange by increasing
8. The turret wobbles
9. Micrometer readout problems
Difficulty exchanging recipient blocks
Colorectal Cancer Family Registry - Coordinating Center Laboratory
University of Southern California, Department of Preventive Medicine
Drafted October 10, 1998
Revised Feb 13, 2004 AD
the tape thickness by adding another layer. This increases the gap between the magnets and the
recipient block, thereby reducing the magnetic force.
Colorectal Cancer Family Registry - Coordinating Center Laboratory
University of Southern California, Department of Preventive Medicine
Drafted October 10, 1998
Revised Feb 13, 2004 AD
v. FREQUENTLY ASKED QUESTIONS
General questions regarding tissue arrays:
1. What about tumor heterogeneity and tissue arrays? How many punches do you need to get a
good representation?
Depends on tissue type and the purpose of the study. As a rule of the thumb, making tissue arrays from normal
breast epithelium, normal kidney and cancers with high degree of regional heterogeneity probably require 2-4
punches per tissue whereas most cancers are well represented with 1-2 punches. See Camp et al, and Torhorst et
al, for technology validation papers discussing the use of tissue arrays in evaluating prognostic markers.
2. How many specimens can you include per array block?
Number of specimens per array depends on the size of the punches and the desired array density. Using 0.6 mm
punches you can construct tissue arrays with 500 or more specimens per block using regular tissue cassettes.
Using 2 mm punches allows construction of tissue arrays with about 50-100 specimens.
3. How long does it take to make tissue arrays?
Making tissue arrays should be considered as a project that involves many steps. Array construction itself is
relatively fast as compared to the other, necessary preparatory steps that include marking of the representative
tissue areas for punching. It is realistic to reserve a couple of weeks for completing tissue array construction
project for an array consisting of 400 specimens. Because setting up an arraying project takes a considerable
amount of time, it often makes sense to construct multiple replicas of the array blocks simultaneously. Making
multiple replicate tissue array blocks is faster and more convenient with the automated arrayer than with the
manual arrayer. See Arrayer comparison table for estimated core transfer rates for the automated and manual
instruments.
4. How do you cut sections from the array block?
Some users prefer to cut sections using regular microtome sectioning techniques; some prefer to use adhesivecoated slides from Instrumedics. The advantage of using coated slides is that even beginners can make good
sections with the system and you have precise control over the orientation of the tissue array sections on the
microscope slides. If you decide to apply conventional sectioning techniques, briefly heating the block at 35-37
degrees C for about 20-30 minutes (and letting the block to cool back to room temperature) before sectioning
improves the section quality.
5. How many sections can you cut from typical tissue arrays?
Depending on the height of the original donor tissue blocks you can cut 100-300 sections from tissue arrays. 150
sections is a reasonable expectation for a typical tissue array.
6. Can you make frozen tissue arrays with Beecher's instruments?
It is possible to make small frozen tissue arrays with the manual instrument. See Fejzo et al for description.
However, this approach is relatively difficult and keeping the tissue array quality high is challenging. Beecher is
currently working on a prototype frozen tissue arrayer device. This instrument is not yet available for sale, but you
can contact us regarding custom arraying.
Questions about the manual arrayer:
7. How to align punches in the manual arrayer?
First, to verify punch alignment, place a blank paraffin block in the block holder and put this complex in position
on the arrayer as if it were a recipient block. Press the smaller punch down until it leaves a mark in the paraffin
surface. Move the turret to switch the larger sampling punch in position, and again make a mark on the paraffin
surface. Adjustment is needed if the marks are not coincidental. Punch holes that appear one in front of the other
need front to back adjustment; holes to the side of each other require left-to-right alignment. The blue arrow
points to the left-to-right setscrews, the orange arrow indicates the location of front-to-back setscrews.
Adjusting front-back alignment of a punch with the setscrew located in front of the arrayer.
Colorectal Cancer Family Registry - Coordinating Center Laboratory
University of Southern California, Department of Preventive Medicine
Drafted October 10, 1998
Revised Feb 13, 2004 AD
Questions arise during the construction of the TMAs:
8. For USC CFR protocol, what percentage of tissue can be taken from one block?
Remove tissue NOT to exhaust the block, so that large section can be cut after all arrays have been made is: about
20% of tissue per block.
9. If there is not enough tissue from a block and a second block from the same subject is available, can the second
block be used to provide more cores for the TMAs?
Yes. Make sure Spec ID be documented.
10. Should tissues from different blocks of the same subject be taken to deposit on the array block?
Ideally and scientifically, same source of tissue should be taken for the same array block, especially for the A/C/N
array which tumor progression can be studied.
11. If a hole in one array block was damaged, what should we do?
Skip that hole which means one replicate block lesser for the set of array blocks. If the tumor is large enough to make
an extra series, these duplicates will consider the QC for the assay. In any mean, always make the same localization
in a set of array blocks for the same donor tumor.
Colorectal Cancer Family Registry - Coordinating Center Laboratory
University of Southern California, Department of Preventive Medicine
Drafted October 10, 1998
Revised Feb 13, 2004 AD
APPENDIX 1.
Protocols for Pathology Reporting and Sample Collection (revised March 1, 2001)
1. All cancer diagnoses should be verified where possible (death certificates, registries, reports, pathology review),
2.
3.
4.
5.
6.
7.
8.
9.
10.
and method of verification should be recorded. Pathology review is the gold standard for verification, and when
possible using the CRFCCS Colorectal Cancer form on http://www.cfr.epi.uci.edu/colon web site.
Tumor blocks should consist of approximately 70-80% tumor cells. If available, a block of normal tissue should
be retained for each patient, particularly if deceased and/or if blood is not available. Preferably, the same tumor
block should be used to obtain sections for MSI (microsatellite instability) and IHC (immunohistochemistry). If
the block containing the highest percent tumor cells contains insufficient tissue for both MSI and IHC sections, a
second tumor block may be used for IHC.
One H&E-stained (hematoxylin and eosin) section representative of the tumor block should be made. If
sufficient material is available, the first and last sections of a block should be stained with H&E.
Protocol for IHC: Tumor tissue only required. Prepare 10 x 5 (or 4) micron paraffin sections from tumor block
and mount on Probe-On slides (no coverslip). Five slides will be used for IHC testing of MLH1, MSH2 and
MSH6 proteins. One H&E slide should be shipped to the IHC testing center along with the five Probe-on slides.
The remaining slides will be stored at the original site as back-up or for future molecular studies. QC for IHC
should include review by an independent observer.
Protocol for MSI: Both tumor and normal tissue required. Prepare either: I) 20 x 5 micron tumor sections on
unbaked slides (no stain/coverslip) where normal and tumor can be isolated separately. or II) 10 x 5 micron
sections from tumor block (containing >80% tumor) and 10 x 5 micron sections from normal block on unbaked,
unstained slides or in separate sterile microcentrifuge tubes. Normal DNA can also be obtained from blood
samples if normal block is unavailable. For sections requiring microdissection, an H&E slide must be prepared
from the same block.
MSI testing is to be performed on all CFRCCS probands and preferably on all colon cancer cases. Where
possible, MSI testing to be performed on two cancers per family, or three when only one of the two gives a
positive result (MSI-H). An endometrial cancer may be used in place of colorectal cancer for MSI testing.
IHC testing for absence of MLH1 and MSH2 (and at some centers MLH6) protein expression should be
performed on all probands and affected relatives with tumors that are MSI positive with greater than 30% of a
minimum of 5 markers, or show MSI with any mononucleotide marker (including: BAT25, BAT26, BAT40 and
BAT34C4). Some centers are funded to test for additional proteins and/or cases.
If blocks need to be returned prior to preparing sections for IHC/MSI, a mini-block containing sufficient tissue
for the required sections may be made and stored.
Following IHC and MSI testing at off-site facilities, all slides (stained, unstained and H&E) will be returned to
the original site for permanent storage.
Slides/sections should be stored in the dark under conditions that insure greatest integrity of the specimen. Both
stained and unstained slides can be stored at room temperature in slide boxes or stacked vertically in slide trays
/cabinets. Storing at 4 C may better maintain protein integrity.
Back to Molecular Characterization & Pathology Main Minutes
Colorectal Cancer Family Registry - Coordinating Center Laboratory
University of Southern California, Department of Preventive Medicine
Drafted October 10, 1998
Revised Feb 13, 2004 AD
APPENDIX 2. Material
Specification of the bar-coded labels for glass slide.
DeltaOne Software 714-528-SCAN
Material Description
TS822
DESCRIPTION:
TS822 label material is designed with an overlaminate flap that is placed over the image after printing.
This “Flap” is for additional image protection against many harsh chemical environments. The adhesive
is
formulated to permanently bond to the most demanding surfaces including textured and contoured
surfaces or in applications where a slightly thicker adhesive is required. This construction is intended for
use in histology, cytology, and research laboratories. The labels are resistant to water, xylene, alcohol,
ethanol, methanol and other common histological stains.
Labels must be applied to the painted or frosted sections of glass slides to insure maximum bond.
CONSTRUCTION:
OVERLAMINATE
Thickness
.002” nominal
Material
clear polyester
Adhesive
.001” nominal clear acrylic
TOP COATING
Thermal transfer receiver coat compatible with hard resin ribbons
BASE MATERIAL
Thickness .
002" nominal
Material
white polyester
ADHESIVE
Thickness
.002" nominal
Material
high holding acrylic
LINER
Thickness
.0032" nominal
Weight 55 lb.
Material
densified, bleached, kraft paper
RIBBON COMPATIBILITY:
Datamax
All except Ovation & E-class series
Zebra
All Models except T402
Eltron
For Desktop Models
Intermec
For Models 3240/3440
1st Choice TR611 TR611Z TR611HZ TR611Z
2nd Choice TR601 TR601Z TR613HZ TR601Z
CHARACTERISTICS:
Service Temperature Range:
-40 F to +257 F (-40 C to +125 C)
Minimum Application Temperature:
+50 F (+10 C)
Typical Minimum Narrow Bar Width: Step Ladder Orientation - .010, Picket Fence Orientation - .005
Using certain printer, ribbon, heat, and speed combinations a smaller narrow bar width may be achieved.
Typical Automatic Applicator/Dispenser Use: Not Tested Poor Fair Good Excellent
Label size and orientation will affect performance. Specific designs must be individually tested.
To assure maximum performance, Sigma Systems materials have been designed and tested for compatibility between
the label stock and thermal transfer ribbon. All Sigma Systems materials are sold as a package, containing label
stock, thermal transfer ribbon and a print head cleaning pen.
All technical information and recommendations are believed to be accurate but do not constitute a guarantee or
warranty. Suitability for any given application is the responsibility of the user. Computype reserves the right to
change specifications without notice.
Colorectal Cancer Family Registry - Coordinating Center Laboratory
University of Southern California, Department of Preventive Medicine
Drafted October 10, 1998
Revised Feb 13, 2004 AD
If you have further questions, please call DeltaOne Software 714-528-7226 . Issue Date: 02/24/00 Revised: 12-18-01
Colorectal Cancer Family Registry - Coordinating Center Laboratory
University of Southern California, Department of Preventive Medicine
Drafted October 10, 1998
Revised Feb 13, 2004 AD
APPENDIX 3.
Shipment Notification Procedure
For Paraffin Embedded Tissue
1. The lab MUST be notified prior to the shipment.
2. Include the following information in your e-mail.

Send to all personnel below:
Ofeliya Avetisyan
E-mail: oavetisy@usc.edu
Tel #: (323) 865-0583
Qingru Feng
E-mail: qfeng@usc.edu
Tel #: (323) 865-0581
Anh Diep
E-mail: atdiep@usc.edu
Tel #: (323) 865-0713

Attach a copy of the standard lab TUMOR SHIPPING LOG sheet.

Provide the FedEx tracking #.
3. Package should be sent to the lab using the following shipping address:
Anh Diep c/o Haile’s Lab
USC Keck School of MED – Norris Cancer Center
Topping Tower 5421, 5427 and 5429
1441 Eastlake Ave.
Los Angeles, CA 90033
Lab Line #: (323) 865-0585
Fax #: (323) 865-0140
4. The lab will acknowledge the arrival of the package immediately upon receiving
(with a “cc” to Pat Harmon). Then follow with a report of the tumor accessing
status within the next few days.
5. After processing (one month turn around time), the lab will be responsible to
return the tissue to the coordinators. We will notify the coordinators in advance
with FEDEX tracking information through e-mail
As soon as the coordinators receive the package, the lab requests a confirmation also through e-mail.
Colorectal Cancer Family Registry - Coordinating Center Laboratory
University of Southern California, Department of Preventive Medicine
Drafted October 10, 1998
Revised Feb 13, 2004 AD
APPENDIX 4.
TMAs SUPPLIES/REAGENTS ORDERING CHECKLIST
Colorectal Cancer Family Registry - Coordinating Center Laboratory
University of Southern California, Department of Preventive Medicine
Drafted October 10, 1998
Revised Feb 13, 2004 AD
APPENDIX 5. Rationales for TMA
The Colorectal Cancer Family Registry (Colon CFR) is an NCI-supported, international consortium, with six
major centers in the U.S., Canada, and Australia (with over 20 participating institutions). The Colon CFR
represents the largest single resource in the world for studies of the causes, prevention, and clinical
management of families at enhanced risk of colorectal cancer. Collectively, to date there are greater than
11,000 families participating in the Colon CFR. The Colon CFR collects blood samples, extensive
questionnaire data on risk factors, and clinical follow-up on participants.
Pathology reports and pathology
specimens are considered a critical core element of the Colon CFR. All centers are required to obtain tumor
tissue and normal margin derived from paraffin-embedded tissue (PET) from probands and selected other
cases in the participating families. Some centers are also collecting fresh frozen tissue to facilitate proteomics
analysis and other analyses that are not feasible from PET samples. In the past, PET samples were simply
sectioned; however, most centers are now either obtaining cores or plan to obtain cores to facilitate tissue
micro-arrays (TMAs), which will provide future scientists with a rich and flexible source of tumor and normal
tissue. Having access to tumor and normal tissue is critical to the success of the Colon CFR, since the ability to
study PET-derived material is an integral part of the overall research plan of the Colon CFR.
Examples of ongoing research that involve pathology specimens include the following:
1. Determination of MSI status on over 8,000 cases, representing the largest study of MSI in the world.
This is a critical variable since evidence suggests that the causes of MSI-H, MSI-L, and MSS tumors
are different from each other, as is the clinical prognosis and response to chemotherapeutic drugs.
2. Conduct of immunohistochemistry (IHC) tests for expression of the mismatch repair (MMR) genes
that cause hereditary non-polyposis colorectal cancer (HNPCC). Studies are underway that are
assessing the agreement between MSI status, IHC results, and MMR mutation status, with results that
will likely change the testing protocols for HNPCC and the MMR genes.
3. Studies of hypermethylation of hMLH, which represents a very important alternative means of
“shutting down” the hMLH1 gene (one of the MMR genes).
4. Studies of somatic mutations in BRAF, which appears to be strongly associated with a CPG island
methylator phenotype (CIMP), an important alternative pathway for causing colorectal cancer.
5. A study to identify the mechanism for the “second hit” in tumors from subjects with a germline
mutation in one of the MMR genes.
6. MSI and IHC results are used to triage who gets tested for germline mutations in the MMR genes,
which will then enable investigators to estimate the lifetime risk of colorectal cancer among mutation
carriers and identify what environmental or lifestyle factors modify this risk. Pathology results are
also used to triage families into new gene linkage studies to map new colorectal cancer genes.
Colorectal Cancer Family Registry - Coordinating Center Laboratory
Drafted October 10, 1998
University of Southern California, Department of Preventive Medicine
Revised Feb 13, 2004 AD
Plans for future research using PET samples include:
1. A study of methylation events in 20 genes in about 8,000 cases, the most comprehensive study
of methylation in colorectal cancer in the world.
2. Greatly expanded studies of somatic mutations, including BRAF, kras, p53, and other genes.
3. Use of pathology variables (MSI and IHC) to triage families into a planned genome-wide
association study.
4. Use of MSI, IHC, and MMR mutation status as key variables in four funded studies of
candidate gene pathways: a) folate/vitamin D/calcium; b) NSAIDs; c) lipid peroxidation; and
d) obesity/metabolic syndrome, IGF).
5. Studies of comparative genome hybridization (CGH) in different types of colorectal cancer
(MSI-H, MSI-L, MSS) and in samples from normal margin compared to adenomatous changes
compared to cancer. Studies of mRNA expression in PET samples are also planned.
In summary, molecular studies of PET samples are critical to the objectives of the Colon CFR to better
understand the causes of colorectal cancer and their biological mechanisms. With this enhanced
understanding, our ultimate goal is to develop more effective preventive strategies, perhaps tailored to each
subject based on their genotypes and the molecular profile of any precursor lesions such as adenomas, and
to develop more targeted therapies.
Colorectal Cancer Family Registry - Coordinating Center Laboratory
University of Southern California, Department of Preventive Medicine
Drafted October 10, 1998
Revised Feb 13, 2004 AD
D. Processing
of
Other types of biospecimens
Colorectal Cancer Family Registry - Coordinating Center Laboratory
University of Southern California, Department of Preventive Medicine
Drafted October 10, 1998
Revised Feb 13, 2004 AD
FINAL
CELLULAR AND GENETIC
MATERIAL
PREPARATION
Colorectal Cancer Family Registry - Coordinating Center Laboratory
University of Southern California, Department of Preventive Medicine
Drafted October 10, 1998
Revised Feb 13, 2004 AD
A. Preparation of DNA
Colorectal Cancer Family Registry - Coordinating Center Laboratory
University of Southern California, Department of Preventive Medicine
Drafted October 10, 1998
Revised Feb 13, 2004 AD
DNA extraction from buffy coat
A. Diep, 9/91; Modifications from F. Baas, H. Bikker, et al.(Hum. Genet. 67, 301(1984),
Gustafson et al. (Analytical Biochem. 165, 294-299 (1987) and Paul et al. (Hum. Genet. 75, 264269 (1984).
The protocol yields DNA of good quality with a standardized procedure
designed to minimize the number of steps and manipulations. To frozen
buffy coat, add lysis buffer (144 mM NH 4Cl, 14 mM NH4HCO3) after
transferring to a 15 ml plastic tube. Mix gently by inversion and let sit for 5
minutes. Add the pellet with pellet buffer (10 mM Tris pH 8.0, 10 mM
EDTA, 150 mM NaCl). To the homogenous suspension, at room
temperature, add proteinase K, 10% SDS, and RNAse (optional) all in the
15 ml tube. Gently invert the tube, and then incubate at 50 C for ~16
hours. The solution should be reasonably clear after proteinase digestion.
The purification is carried out by adding protein-denaturant buffers, phenol
and chloroform. Mix well. Separate phases by centrifuging at 3500 rpm for
15 minutes. Remove the bottom phase and discard. Check and repeat the
extraction until all particles have gone to the interphase, leaving a clear
DNA aqueous phase. Precipitate by adding NaCl to make a final
concentration of 100-250 mM and 2 volumes of cold Ethanol. Mix to obtain
a tight white precipitate. Cold spin at 3500 rpm for 30 minutes. Aspirate the
supernatant . Air dry the pellet. Resuspend it in TE (10 mM Tris, 0.1 mM
EDTA, pH 8.0). Mix the tube until the DNA is completely resuspended. After
the DNA is completely dissolved, remove some aliquots to determine its
purity, concentration, integrity, and digestibility. Store the working DNA
solution at 4C and the back ups at -20C.
Colorectal Cancer Family Registry - Coordinating Center Laboratory
University of Southern California, Department of Preventive Medicine
Colorectal Cancer Family Registry - Coordinating Center Laboratory
University of Southern California, Department of Preventive Medicine
Drafted Jan. 11, 1999
Revised Feb 11, 1999 AD
Drafted October 10, 1998
Revised Feb 13, 2004 AD
STEPWISE PROCEDURE
Protocol designed to have:
 The information of when a step can be interrupted. Condition on hold.
 Volume for the aliquots prepared.
Reagents and Equipment
-20C, -80C freezers
0.6x TBE, pH 8
10 ml tube racks
10% SDS (w/v)
10 x Ficoll loading buffer
12x75 mm borosilicate disposable culture tubes
37C, 50C water bath
4C refrigerator
5 M NaCl
Aqueous alcohol (70%, v/v)
Biohazard waste container
Black sharpie pen
Blood protective pads
Blood protective pads
Centrifuge
Chloroform
Cold absolute ethanol
Compet dispenser
Sterile 10 ml disposable pipettes
ddH2O
Disinfectant: Bleach solution, 0.1% phenol
Disposable latex gloves
Electrophoresis apparatus
Ethidium bromide (5 mg/ml)
Fluorometer
Foil
Fume hoods
Glass beakers waste
Glass bottle for organic waste
Glass waste container
Hind III and 10X reaction buffer
Hoechst dye
Ice bucket
Isoamyl alcohol
Colorectal Cancer Family Registry - Coordinating Center Laboratory
University of Southern California, Department of Preventive Medicine
Colorectal Cancer Family Registry - Coordinating Center Laboratory
University of Southern California, Department of Preventive Medicine
Drafted Jan. 11, 1999
Revised Feb 11, 1999 AD
Drafted October 10, 1998
Revised Feb 13, 2004 AD
Kimwipes
Lysis buffer
Mixer
Molecular biology grade agarose
MW markers
P20, P200, P1000 Pipettemen
Parafilm
Pellet buffer
pH meter: HCl, NaOH
Phenol
Pipetaid
Proteinase K
RNase A
Spectrophotometer
Sterile aerosol-preventive pipette tips
Sterile polypropylene 15 ml centrifuge tubes
Stirrer
TE, pH 8
Transfer pipettes
Solution Preparation:
Lysis Buffer (500 ml)
0.144 M NH4Cl [MW=53.49]
0.0144 M NH4HCO3 [MW=79.06]
Raise to 500 ml with sterile ddH20.
Filter-sterilize.
3.851 g
0.553 g
5M NaCl (500 ml)
146.1 g NaCl, raise to 500 ml with sterile ddH20.
Pellet Buffer
10 mM Tris-HCl, pH 8.0
10 mM EDTA
150 mM NaCl
Stock Solutions:
1M Tris-HCl, pH 8.0
0.5M EDTA
5M NaCl
Sterile ddH2O
Total volume
0.5 ml
1.0 ml
1.5 ml
47.0 ml
50.0 ml
Colorectal Cancer Family Registry - Coordinating Center Laboratory
University of Southern California, Department of Preventive Medicine
Colorectal Cancer Family Registry - Coordinating Center Laboratory
University of Southern California, Department of Preventive Medicine
1.0 ml
2.0 ml
3.0 ml
94.0 ml
100.00 ml
Drafted Jan. 11, 1999
Revised Feb 4, 1999 AD
Drafted October 10, 1998
Revised Feb 13, 2004 AD
TE, pH 8.0
10 mM Tris-HCl, pH 8.0
1 mM EDTA, pH 8.0
Stock Solutions:
1M Tris-HCl, pH 8.0
5 ml
0.5M EDTA
1 ml
Raise to 500 ml with sterile ddH20. Autoclaved.
Phenol Preparation
1. Melt phenol by heating @ 650 C.
2. Add 1 volume of 0.5M Tris pH 8.0. Shake vigorously.
3. Separate by using separatory funnel. Discard aqueous layer ( top layer ).
4. Repeat step 2 again.
5. Check pH of both phenol and aqueous phase :
pH of phenol 6.5 - 7.0; pH of aqueous phenol.
6. Separate phases.
7. Transfer phenol to brown bottle and add 1/3 volume of TE.
8. Store at 40C
Proteinase K
A frozen stock is prepared by dissolving Proteinase K lyophilized powder at 10 mg/ml in
dissolving solution as instructed by the manufacturer. Once dissolved, the solution is
aliquotted into smaller volume. Store at -20 oC. The enzyme is stable at least one year.
RNase A
Frozen stock of 5 units/l is prepared by dissolving 1 g of RNase lyophilized powder in
sufficient sterile water to give 5 units of RNase/l, followed by boiling 15 minutes to
inactivate DNases. The stock is cooled, make 1 ml aliquots into sterile 2 ml tubes, and
stored frozen at -20 oC.
Hoechst dye Solution (“ fluorometry solution””)
Add 12 g NaH2PO4 ( monobasic, anhydrous) or 13.8 g NaH2PO4.H2O, 233.8 g NaCl, and
4 ml of 500 ug/ml Hoechst dye. Raise volume to 2 liters with ddH2O.
Adjust pH to 7.4 with 10 N NaOH (~20 ml).
Store at -20C in 1 ml aliquots using brown or foil-wrapped bottles or tubes.
Solution can be stored at -20C up to 6 months.
10 x TBE, pH 8 ( 1 L)
90 mM Tris-HCl, pH 8.3 [FW=121.1]
108 g
90 mM Boric acid
55 g
2 mM EDTA
40 ml 0.5 M EDTA (or 7.44 g EDTA [FW=372.2] )
Raise to 1 L with sterile ddH20.
Colorectal Cancer Family Registry - Coordinating Center Laboratory
University of Southern California, Department of Preventive Medicine
Colorectal Cancer Family Registry - Coordinating Center Laboratory
University of Southern California, Department of Preventive Medicine
Drafted Jan. 11, 1999
Revised Feb 11, 1999 AD
Drafted October 10, 1998
Revised Feb 13, 2004 AD
250 ml Nuclei Lysis Mixture: (freshly prepared)
In wash bottle, add 235 ml pellet buffer
12.5 ml 10% SDS
2.5 ml 10 mg/ml Proteinase K
1L
Phenol:
25:
500 ml
Chloroform:
24:
480 ml
Isoamyl alcohol
1
20 ml
Stepwise Procedure:
Isolation of Nuclei:
1. Thaw the frozen pellets. Mix 5-7 times by gentle inversion.
2. Transfer the pellet from 2-ml microcentrifuge tube into the labeled sterile 15 ml
polypropylene centrifuge tube.
Wipe top of tubes with ethanol before opening the cap.
Do not use glass tube, because DNA will stick to glassware.
Polypropylene tubes must be used, because chloroform which is used later in the organic
extraction is a solvent for polystyrene.
3. Rinse the tube with a equal volume of lysis buffer. Let the tube sit on ice for 5 minutes
to disrupt cell membrane.
!!!Freeze at -80C until further extraction.
4. Thaw the frozen pellets by placing the 15 ml tube in room temperature water bath.
5. When thawed, resuspend in 3 times the original volume of pellet buffer.
For large scale setup, bring all tubes to same final volume with pellet buffer.
6. Keep the tube on ice for at least 5 minutes to complete lysis of the nuclei membrane.
Invert at 20-30 second interval several times.
!!!Store at 4C until further extraction.
Dissociation of nucleic acid-protein complexes
7. Add Proteinase K to a 200 g/ml final concentration.
Optimal proteolytic activity need at least 100 ug of Protinase K per 10 cells.
There is ~5x106 cells in 1 ml whole blood; ~20 ml whole blood collected from this study.
8. Add 10% SDS (w/v) to a 1% final concentration. Mix gently by inversion. Solution is
now viscous as DNA is released from cells.
For large scale setup, SDS and Proteinase K can add together with pellet buffer in one
step by making nuclei lysis mix. Again, bring all tubes to the same final volume.
9. Incubate tube in a 50C waterbath overnight (16 hours) without agitation.
RNA removal (optional):
RNase may be added at this time, though generally not necessary.
10. Add RNase A to a concentration of 100 g/ml.
11. Incubate at 370 C for 30 minutes. Spin to collect volume.
Colorectal Cancer Family Registry - Coordinating Center Laboratory
University of Southern California, Department of Preventive Medicine
Colorectal Cancer Family Registry - Coordinating Center Laboratory
University of Southern California, Department of Preventive Medicine
Drafted Jan. 11, 1999
Revised Feb 12, 1999 AD
Drafted October 10, 1998
Revised Feb 13, 2004 AD
Organic Extraction to deproteinize DNA:
12. First extraction, add equal volume of phenol only. Mix gently until solution forms an
milky emulsion.
13. Centrifuge at room temperature at 3500 rpm for 15 minutes. Remove phenol, lower
phase by sterile transfer pipette. Discard phenol.
The white precipitates at the bottom of the top interphase layer are proteins and are to be
extracted out from the DNA solution. An extensive white film indicates incomplete
digestion with Proteinase K. More digestion should be performed.
Do not use glass pipette, because DNA will stick to glassware.
DNA should not be left in the presence of denaturants.
14. Second extraction, add equal volume of the mixture of denaturing agents, 25:24:1
phenol: chloroform: Isoamyl. Mix gently until solution is milky.
15. Centrifuge at room temperature at 3500 rpm for 15 minutes. Remove the mixture of
denaturing agents, lower phase by sterile transfer pipette. Discard.
16. Add equal volume of chloroform only. Mix thoroughly. Spin at 3500 rpm for 15
minutes. Remove lower phase, chloroform to be discarded. At this step, carefully work tip
around the tube and get as much as you can the white film of precipitates at the bottom of
the top layer without disturbing the clear DNA layer on the top.
Keep bottom layers of phenol and chloroform in case DNA is fail to be obtained in the
top layer.
Precipitation of DNA:
17. Add 5 M NaCl to a final concentration of 100-250 mM NaCl.
18. Add 2 volumes of cold absolute ethanol. Mix immediately by inverting the tube for
at least 30 seconds. Observe for the formation of a visible mass of cloudy, white threadlike strands of DNA.
If no precipitate is seen because DNA is fragmented, freeze the sample at -80C for 30
minutes to pellet the DNA before centrifugation.
!!!Freeze at -80C until further extraction.
19. Centrifuge at room temperature at 3500 rpm for 30 minutes. The DNA will be visible
as a small white pellet at the bottom of the tube.
20. Decant the supernatant by invert the tube. The DNA pellet is very loose at this point
and care must be used to avoid losing the pellet.
21. Wash the DNA pellet and the sides of the tube with 2 ml of 70% ethanol for several
times. Centrifuge at room temperature at 3500 rpm for 1 minute. (optional)
22. Remove most of the supernatant by invert the tube upside down on a clean absorbent
paper for about 5 minutes. Do not leave tube in this position for too long, because the
pellet will drop down on the paper.
23. Continue to air dry the pellet thoroughly for 30 minutes.
The pellet should not be overdried. Over dehydration of DNA can cause denaturation of
the DNA.
!!!Store at 4C until further extraction.
Colorectal Cancer Family Registry - Coordinating Center Laboratory
University of Southern California, Department of Preventive Medicine
Colorectal Cancer Family Registry - Coordinating Center Laboratory
University of Southern California, Department of Preventive Medicine
Drafted Jan. 11, 1999
Revised Feb 12, 1999 AD
Drafted October 10, 1998
Revised Feb 13, 2004 AD
Rehydration of DNA:
24. Resuspend the dry pellet in 1-1.5 ml 1xTE by gently invert tube at 370C for 4 hours or
room temperature for overnight. The DNA will continue to dissolve at 40C for 1-2 days.
Make sure the TE volume added sufficient to rehydrate the pellet, at the same time try to
obtain a high concentration DNA.
Make certain that the DNA is homogeneous before the determination of its concentration.
Although viscous, the solutions should look homogeneous with no large “ globs” of
partially undissolved DNA.
Average yield for genomic DNA from whole blood or buffy coat is 25-40 g/ ml of blood.
!!!Store working solutions at 4C and back ups at -20C.
Quantitation of DNA by OD260 determination:
 Prepare a series of dilution of small aliquots of the DNA in ddH2O in labeled Ubottom microtitrate plate. (Usually 1, 2, and 5 l to a final volume of 100 l). Mix
diluted samples well with pipetteman.
 A blank must always be used to zero the Spectrophotometer. This is the same ddH2O
used for diluting DNA.
Using the Beckman DU Series 600 Spectrophotometer:
1. From the main menu, click A:\Nucleic Acid from the Custom Applications list.
2. The next screen should have three columns, OD260nm, OD280nm and
OD260/OD280. Make sure “UV on” near the bottom of the screen.
3. Insert the blank, and click on “Blank” on the bottom left of the screen.
4. Insert your samples and click on “ReadSamples” on the top left of the screen
after each sample. Clean the cuvette with water and alcohol. Invert and tap
onto paper towels to dry the cuvette as much as possible. Dry the outside of the
cuvette carefully with Kimwipes or lens paper.
5. When you have gone through all your samples, turn off UV. Then click on
“SaveClear” . Click on “A:\” to toggle between A drive (in computer) and B
drive (disk). Click on “ Work_Res” to change the name of the file. Click on
“QUIT” to exit. It will ask you if you want to save changes in method A:\
Nucleic Acid and you should click “YES” .
6. From the main screen, choose “FILE UTILITIES” under the Utilities list.
7. Click on “Disk status” . In B:\FIX_DATA there should be B:\filename. DUF
where filename is what you re-named “ Work_Res” in step 5. Click on this
filename and then click on “Convert” . Choose destination drive [B] by
clicking on [A]. Choose what kind of file by toggling between [LOTUS] and
[ASCII]. (Haile’ s lab has Lotus.) Then click on “OK” . “Quit” to exit.
After bringing the disk to your computer, open Lotus 1-2-3 and then open the
disk. Under “ Lotus” have the computer look for files with the ending “WKS”
by changing “WG?” Try to save the file as an excel file so you can open Excel
and calculate the OD260 / OD280 Ratio andDNA Concentration in ng/ul.
OD260* dilution factor * coefficient for DNA
Where dilution factor is usually 100, 50 and 20, and the coefficient for DNA is 50.
Colorectal Cancer Family Registry - Coordinating Center Laboratory
University of Southern California, Department of Preventive Medicine
Colorectal Cancer Family Registry - Coordinating Center Laboratory
University of Southern California, Department of Preventive Medicine
Drafted Jan. 11, 1999
Revised Feb 12, 1999 AD
Drafted October 10, 1998
Revised Feb 13, 2004 AD
Quantitation of DNA using fluorometer:
 For each sample, make 10 ul DNA solution in 2 ml Hoechst dye solution. Vortex 30
seconds to mix. Let sit at least 15-20 minutes for dye reaction.
For the same batch of measurement, make sure that all samples will have the same
amount of reaction duration.
 Use 12x75 mm borosilicate disposable culture tubes.
 Turn on fluorometer, allow 15 minutes to warm up.
 Set GAIN to 10 and SPAN all the way counter clockwise. Adjust blank to zero by
turning the ZERO knob until span reading indicates 000.
 Read samples and record.
 If reading is higher than 2000 ug/ml or above the linear range, dilute DNA 3:1 with
dye solution and read again.
 Blank using 2 ml Hoechst dye solution.
 Wipe tube with kimwipes or lens paper.
Using the Sequoia-Turner M450 fluorometer:
1. The standard curve is made using 0-2000 ug/ml calf thymus DNA with stock
concentration of 100 ng/ul in 1 ug/ml Hoechst dye solution.
3. Plotting mean span vs. concentration of standard curve:
Prepare DNA standards in duplicate, as listed in the table below:
Concentration,
[ ___ ng/200 ul]
Volume of 100 ng/ul
stock DNA to use, in ul
0
50
100
200
300
500
1000
2000
0
0.50
1.00
2.00
3.00
5.00
10.00
20.00
Volume of
fluorometry solution
to use, in ul
2000.0
1999.5
1999
1998
1997
1995
1990
1980
Make sure the standard curve is in linear range.
*A new standard curve should be prepared for each new batch of Hoechst dye
solution.
Determination of DNA purity by taking OD260/OD280 ratio:
The quality of the DNA sample isolated will be evaluated before use by checking its
absorption spectra.
Calculate the ratio of absorbance at 260 to 280 nm. Ratio should be optimal from 1.8-2.0.
Very low ratio (<1.6) may indicate significant protein contamination.
Very high ratio (>2.0) may indicate significant RNA contamination.
Samples that have consistently low or high ratios may require re-extraction; increase the
sample incubation time with Proteinase K; more ethanol washes to remove trace phenol.
Colorectal Cancer Family Registry - Coordinating Center Laboratory
University of Southern California, Department of Preventive Medicine
Colorectal Cancer Family Registry - Coordinating Center Laboratory
University of Southern California, Department of Preventive Medicine
Drafted Jan. 11, 1999
Revised Feb 12, 1999 AD
Drafted October 10, 1998
Revised Feb 13, 2004 AD
Determination of DNA Integrity:
Run uncut DNA on a 1% agarose gel with a standard of known concentration.
Observe for the intact band of DNA at high molecular weight and no sign of degradation
shown as a smear near the lowering part of the gel.
Precautions should be taken to maximize genomic DNA molecular weight by minimizing
shearing force due to not gentle mixing during extraction steps.
Visually check DNA purity by observing no sign of RNA which migrate at very low
molecular weight.
Determination of DNA Digestibility:
The absence of trace organic solvents, salts, cellular proteins and proteinase K in the
final DNA solution is important for susceptibility of the genomic DNA to restriction
enzyme action.
 Cut 200 ng of isolated DNA with 1 to 5 units of Hind III in the presence of 10X
reaction buffer, and appropriate cocktail for the enzyme.
 Incubate overnight at 37 oC .
 Electrophoresis run the digested product and same amount of undigested DNA on 1%
agarose in 0.6x TBE, then UV visualizing the ethidium bromide stained material to
examine its digestibility.
Samples digestible with these restriction enzymes will appear as an even smear on the
gel and have no sign of high molecular weight bands.
Repeat digestion of any samples that were not digestible with either restriction
enzymes. If sample is consistently indigestible, it may require re-extraction or
replacement. Uncut samples that are severely degraded require replacement also.
DNA Typing for mix-up clarification by microsatellite analyses :
Use microsatellite that has high discriminating power to QA&QC test of a small number
of randomly picked DNA samples. Microsatellite analysis allows us to compare the
genotypes between aqueous DNA source and DNA from bloodstain.
PCR setup of less than 20 ng of DNA using D1S80 that is mapped at the distal of
Chromosome 1 and has the discriminating power of 95-98%. PCR product of 350 bp-1 kb
size will be electrophoresed on native agarose gel and then UV-visualize the genotype.
Samples that are identified as mix-up will be excluded or hold for further resolution.
Data Records and Reports
Extraction Sheet
Concentration Sheet
Digestion Sheet
DNA Typing Sheet
DNA inventory log-in
Colorectal Cancer Family Registry - Coordinating Center Laboratory
University of Southern California, Department of Preventive Medicine
Colorectal Cancer Family Registry - Coordinating Center Laboratory
University of Southern California, Department of Preventive Medicine
Drafted Jan. 11, 1999
Revised Feb 4, 1999 AD
Drafted October 10, 1998
Revised Feb 13, 2004 AD
DNA extraction from dry blood stains
Elkhouly, Liu et al.(Haile’s 3/2000), modified from Brockmoeller et al., and Lin’s
Reagents and Equipment:
1.2 mm-HARRIS MICRO-PUNCH
Micro-titrate plate, V-bottom
5% Chelex
ddH2O
Stir bar
Stirrer
Glass beaker
P20, P200 Pipettemen
Sterile aerosol-preventive tips
Disposable latex gloves
Glass waste container
Kimwipes
01% phenol
sterilized toothpick
Solution Preparation:
5% Chelex
Add 5g of Chelex 100 Resin in 100 ml ddH2O
STEPWISE PROCEDURE
Making blood spot:
1. Place the bloodstain onto the cutting mat.
2. Gently push the 1.2mm-HARRIS MICRO-PUNCH downward onto the sample,
rotating the barrel clockwise and counter-clockwise.
3. Lift the barrel up from the sample.
4. Transfer the spot over a micro-titrate well, push it out by pressing the plunger, release
it with a sterilized toothpick.
5. Decontaminate the hole-puncher with 0.1% phenol between samples.
Extraction: To each micro-titrate well with a blood spot,
1. Add 10ul of methanol. Air dry thoroughly.
2. Add 10ul BSA to the final concentration of 2ng/ul.
3. Heat for 20 minutes at 95C on a thermal cycler.
4. The sample is ready to use for PCR reaction (suggested volume is10 ul).
Colorectal Cancer Family Registry - Coordinating Center Laboratory
University of Southern California, Department of Preventive Medicine
Colorectal Cancer Family Registry - Coordinating Center Laboratory
University of Southern California, Department of Preventive Medicine
Drafted Jan. 11, 1999
Revised Mar 14, 2000 AD
Drafted October 10, 1998
Revised Feb 13, 2004 AD
DNA extraction from paraffin-embedded-tissue
(USING THE QIAamp DNA MINI KIT)
Notes: - Use PCR-grade water and reagents
- Use filter tips
- Ensure that buffer AL, buffer AW, and proteinase K have been prepared according
to instructions (see below). If a precipitate has formed in buffer AL or buffer ATL,
dissolve by incubating at 55oC for 2 min
- Prepare a 55oC water bath for use in step 7, and a 70oC water bath for use in steps 9
and 16
- Equilibrate buffer AE or water to 70oC for elution in step 16
- All centrifugation is carried out at room temperature
To the buccal cell pellet add 180ml buffer ATL
Add 20ml of proteinase K stock solution, mix by vortexing, and incubate at 55oC for 3
hours. Vortex occasionally during incubation to disperse the sample
Add 200ml buffer AL to the sample, mix again thoroughly by votexing, and incubate at
70oC for 30min
Note: A white precipitate may form on addition of buffer AL. In most cases it will dissolve
during incubation at 70oC. The precipitate does not interfere with the QIAamp procedure,
or with any subsequent application.
Add 200ml of ethanol (96-100%) to the sample and mix thoroughly by vortexing.
Note: A white precipitate may form on addition of ethanol. It is essential to apply all of
the precipitate to the QIAamp spin column. This precipitate does not interfere with the
QIAamp procedure, or with any subsequent application.
Place QIAamp spin column in a 2ml collection tube (provided). Carefully apply the mixture
from step 10 (including the precipitate) to the QIAamp spin column without moistening
the rim, close the cap, and centrifuge at 6000 x g (8000rpm) for 1min.
Note: If the lysate has not completely passed through the column after centrifugation,
centrifuge again at higher speed until the QIAamp spin column is empty.
Place the QIAamp sping column in a clean 2ml collection tube (provided) and discard the
tube containing the filtrate.
Carefully open the QIAamp spin column and add 500ml buffer AW1. Centrifuge at 6000 x g
(8000rpm) for 1min. Place the QIAamp spin column in a clean 2ml collection tube
(provided) and discard the collection tube containing the filtrate.
Carefully open the QIAamp spin column and add 500ml of buffer AW2. Centrifuge at full
speed for 3min.
Colorectal Cancer Family Registry - Coordinating Center Laboratory
University of Southern California, Department of Preventive Medicine
Drafted Jan. 11, 1999
Revised Mar 14, 2000 AD
The additional full speed spin removes all traces of buffer AW from the QIAamp spin
Colorectal Cancer Family Registry - Coordinating Center Laboratory
University of Southern California, Department of Preventive Medicine
Drafted October 10, 1998
Revised Feb 13, 2004 AD
column before elution.
Place the QIAamp spin column in a clean 1.7ml microfuge tube (not provided) and discard
the collection tube containing the filtrate.
16. Carefully open the QIAamp spin column. Elute the DNA once with 50ml buffer AE.
Incubate at room temperature for 1min, then centrifuge at full speed for 1min.
Reagents and Solutions:
QIAamp DNA Mini Kit (QIAGEN Cat. # 51304 (50 preps), Cat. # 51306 (250 preps))
Proteinase K stock solution: Add 1.4ml of distilled water to the lyophilized QIAGEN
proteinase K. Store at –20oC.
Buffer AL: Prepare buffer AL by decanting all of reagent AL1 into buffer AL (Reagent
AL2). Mix thoroughly by shaking. Buffer AL is stable for at least 1 year when stored in the
dark at room temperature.
Do not add QIAGEN proteinase K directly to buffer AL
Buffer AW: Buffer AW is supplied as a concentrate. Before using for the first time, add
the appropriate amount of ethanol (96-100%) to buffer AW concentrate as indicated on
the bottle. Buffer AW is stable for at least 1 year when stored closed at room
temperature.
Colorectal Cancer Family Registry - Coordinating Center Laboratory
University of Southern California, Department of Preventive Medicine
Colorectal Cancer Family Registry - Coordinating Center Laboratory
University of Southern California, Department of Preventive Medicine
Drafted Jan. 11, 1999
Revised Mar 14, 2000 AD
Drafted October 10, 1998
Revised Feb 13, 2004 AD
B. Preparation of RNA
(To Come)
Colorectal Cancer Family Registry - Coordinating Center Laboratory
University of Southern California, Department of Preventive Medicine
Drafted October 10, 1998
Revised Feb 13, 2004 AD
C. Preparation
of
Lymphobastoid cell line
(To Come)
Colorectal Cancer Family Registry - Coordinating Center Laboratory
University of Southern California, Department of Preventive Medicine
Drafted October 10, 1998
Revised Feb 13, 2004 AD
STORAGE,
PACKING AND SHIPPING
OF
BIOLOGICAL SPECIMENS
Colorectal Cancer Family Registry - Coordinating Center Laboratory
University of Southern California, Department of Preventive Medicine
Drafted October 10, 1998
Revised Feb 13, 2004 AD
1. Storage
A number of technical issues may limit the usefulness of the biospecimens stored for many
years in a biological bank:
 The temperature of storage, which affects stability.
 The availability of the appropriate biological material needed for a given analysis.
 The amount of the blood samples recruited per subject which may limit the number of
the laboratory analyses
 The size of each aliquot should be the smallest increment possible, to avoid thawing
and re-freezing the samples, a process which may damage some analytes.
In this section, we documented the methods of storage for different cellular and genetic
material preserved from 30cc to 40cc blood of each subject, as well as the availability of the
resources and the size of the aliquots for this study:
A. Blood cards
a. Each participant will have one blood card with 22 x 20ul-blood spots.
b. This card will be wrapped with a weighing paper and put in a coin envelope.
c. File the envelope in a banker box chronologically.
d. Store at room temperature in a climate controlled laboratory.
The capacity of a 2-drawer banker box: about 150 envelops per drawer.
B. Plasma
a. Each participant will have 20-30 of 0.5 ml aliquots of plasma from 30 ml blood.
b. The number of aliquots obtained will be split into two batches per subjects.
c. Store in two different -70 oC freezers.
C. Buffy coat/Red Blood Cell Residual from EDTA blood
a. Transfer buffy coat/blood residual into 3-15ml centrifuge tubes respectively.
b. The three buffy coat/blood residual tubes will be temporarily stored at -20 oC for DNA
extraction.
c. Extract the DNA from these tubes in two batches. First extraction batch will only be for
blood tube #1. The others will be extracted in second batch.
D. WBCs from ACD blood
a. Each participant will have about 4 of the 1 ml aliquots of WBCs with less than 2x106
cells per aliquot.
b. Store in two different liquid nitrogen tanks.
E. DNA
a. From blood spots: DNA extracted from this source is for direct PCR only, no storage
required.
b. From EDTA buffy coat/ residual rbc or EBV cell lines: All extracted DNA from
each subject will be stored in two different -70 oC freezer. Preserve DNA in high
concentration allows us to provide the researchers with different concentrations
according to their studies. However, at least one 10-g aliquot of original stock must be
reserved, until a cell line has established.
Colorectal Cancer Family Registry - Coordinating Center Laboratory
University of Southern California, Department of Preventive Medicine
Drafted Jan. 11, 1999
Revised Dec 30, 2002 AD
2. Packing
Colorectal Cancer Family Registry - Coordinating Center Laboratory
Drafted October 10, 1998
University of Southern California, Department of Preventive Medicine
Revised Feb 13, 2004 AD
A. Specimen preparation for packing
1) Allow samples to thaw if aliquots need to be made. Aliquot the amount of
sample requested into the corresponding size of tubes.
2) Check that all the caps are tightened.
3) Check all tubes or specimens in general against the shipping list.
B. Packing
1) If only a few samples are shipped:
a. Wrap tubes onto cardboard panels and in bubble wrap.
b. Place in brown cardboard shipping boxes.
2) If many samples are shipped:
a. Place individual sample in storage boxes or mailers.
b. Secure the specimens with sufficient fillers.
c. Secure storage boxes with rubber bands.
d. Wrap in bubble wrap.
e. Place storage boxes in cardboard shipping boxes.
f. Again secure the storage boxes with sufficient fillers.
3) Packing the blood cards:
a. Separate the blood cards from different subject by placing each card in
an envelope.
b. Pack all blood cards in a ziplock bag or cardboard box with sufficient
desiccants (calcium sulfate, CaSO4 ).
4) Surrounding the plasma container with sufficient amount of dry ice.
5) Pack the DNA with sufficient amount of ice pack. Otherwise, dry down the
DNA.
3. Shipping
A. Shipping condition:
1) Blood cards: at room temperature.
2) Plasma: at -20oC. Samples should remain frozen at all time.
3) DNA: cold at ~ 4oC. Dry DNA pellets can be shipped at room
temperature.
4) WBCs: at least under -20oC. Samples should remain frozen at all
time.
B. Shipping:
1) Include in the box the following documentation:
a. Shipping information sheet
b. Receiving confirmation sheet (with condition-on-arrival filled out)
c. Special handling instruction
d. Biohazard warning statement
Colorectal Cancer Family Registry - Coordinating Center Laboratory
University of Southern California, Department of Preventive Medicine
Drafted Jan. 11, 1999
Revised Mar 14, 2000 AD
Colorectal Cancer Family Registry - Coordinating Center Laboratory
Drafted October 10, 1998
University of Southern California, Department of Preventive Medicine
Revised Feb 13, 2004 AD
2) Make sure the following information will be included on the outside of the
shipping box:
a. Mailing address of recipient:
b. Brightly colored self-adhesive warning label:
DO NOT DELAY - BIOLOGICALS
PACK IN________________(DRY ICE, ICE PACK)
IMMEDIATELY ________________(FREEZE AT -70C) UPON ARRIVAL
3) For international packages:
a. Use the warning label in the language of the recipient country
b. Place shipper’s certifications in plastic envelops on the outside of the
package
c. The shipper’s certification letter will state clearly the shipping content and
they are not restricted
4) All domestic shipments are sent via ___with a guaranteed second day delivery.
5) Log in the tracking number and monitor the progress of the
shipment.
Always ship the biospecimens of any subject in two batch!
Colorectal Cancer Family Registry - Coordinating Center Laboratory
University of Southern California, Department of Preventive Medicine
Drafted Jan. 11, 1999
Revised Mar 14, 2000 AD
Colorectal Cancer Family Registry - Coordinating Center Laboratory
Drafted October 10, 1998
University of Southern California, Department of Preventive Medicine
Revised Feb 13, 2004 AD
SPECIMENS DATABASE
Colorectal Cancer Family Registry - Coordinating Center Laboratory
Drafted October 10, 1998
University of Southern California, Department of Preventive Medicine
Revised Feb 13, 2004 AD
Scheme for specimen ID
Each sample is assigned a study ID number when the subject is recruited by the
coordinating center. The study ID will appear only on the database but not on any label.
All tubes and blood cards will have a barcode label with seven digits followed by one
check digit for a total of eight digits. Each label will have the letters “CFR” to indicate
the sample belongs to this study. The seven digit numbering system will begin with
0000001 and follow sequentially. Each label will have a different barcode number. The
check digit is a calculated number that is a function of the other seven numbers of the
barcode. The purpose of the check digit is to verify the correct barcode number is being
typed during data entry.
Additionally, each specimen will have more specific information about it built into a
series of Access 97 database tables. The entire Access 97 database is called “CFR” and
the following list details the 13 tables within this CFR database.
(list)
These tables will all be linked together by the study ID (or the barcode?).
Colorectal Cancer Family Registry - Coordinating Center Laboratory
University of Southern California, Department of Preventive Medicine
Drafted October 10, 1998
Revised Dec 30, 2002 AD
Blood cards
Colorectal Cancer Family Registry - Coordinating Center Laboratory
University of Southern California, Department of Preventive Medicine
Drafted October 10, 1998
Revised Feb 13, 2004 AD
A bar code will be given to every envelope of 2 blood cards.
Plasma
A bar code will be given to each tube.
Buffy coat from EDTA blood
A bar code will be given to each tube.
WBCs from ACD blood
A bar code will be given to each tube.
Residual cells from both ACD and EDTA blood
A bar code will be given to each tube.
Paraffin-embedded-tissue slides
A bar code will not be given.
Paraffin-embedded-tissue section in the tube
A bar code will be given to each tube.
Frozen tissue
???
DNA
A bar code will be given to each tube.
RNA
A bar code will be given to each tube.
EBV cell lines
???
Colorectal Cancer Family Registry - Coordinating Center Laboratory
University of Southern California, Department of Preventive Medicine
Colorectal Cancer Family Registry - Coordinating Center Laboratory
University of Southern California, Department of Preventive Medicine
Drafted October 10, 1998
Revised Sept. 9, 1999 AD
Drafted October 10, 1998
Revised Feb 13, 2004 AD
Scheme for location assignment of biospecimens
Aliquots from each specimen will be split into two batches, which will be stored in two
different freezers. This will ensure that one set of aliquots will be unharmed in case of a
power failure.
Blood cards
Each card will have a location assignment such as 0001-01:
1. first 4 digits assigned for the subdivision, only file 10 cards per a subdivision,
2. next 2 digits identified the order where the card will be placed.
Plasma
Buffy coat from EDTA blood
WBCs from ACD blood
Residual cells from both ACD and EDTA blood
Paraffin-embedded-tissue slides
Each slide will have a location assignment such as 0001-01:
1. The first 4 digits are assigned for each individual block, it is also the subdivision
number of the system.
2. The next 2 digits will identify the order where the slide will be placed in that
subdivision.
Frozen tissue
DNA
RNA
EBV cell lines
Colorectal Cancer Family Registry - Coordinating Center Laboratory
University of Southern California, Department of Preventive Medicine
Colorectal Cancer Family Registry - Coordinating Center Laboratory
University of Southern California, Department of Preventive Medicine
Drafted October 10, 1998
Revised Sept. 9, 1999 AD
Drafted October 10, 1998
Revised Feb 13, 2004 AD
Guidelines for labeling specimens
1. make sure the blood tube labeling information corresponds to blood ticket provided
by the USC coordinating center upon receiving.
2. use a permanent marker to label if a printout is not supplied.
3. paraffin-embedded slides should be labeled with Superfrost Marker as any other ink
will be removed in the xylene. Slides should never have barcode labels on them.
4. pre-label all materials (tubes or slides) before working with the specimens.
5. place the 8 digit barcode on the side of the tube.
6. label the 7 digit alphanumeric code on the cap of the tube.
7. cryogenically approved labels should be used with ink that is stable under freezing
condition.
Thus, each specimen will require about 17 study ID printouts:
1 for blood log sheet
1 for the envelope of the blood cards
2 for blood cards
5 for QC assays
8 for DNA/RNA isolation/lymphoblastoid cell line setup
Each specimen also will require about 36 barcode labels:
20 to 24 for plasma
3 to 5 for EDTA buffy coat
10 for Ficoll-separated WBC aliquots
1 for residual cells from both EDTA and ACD tubes
Each specimen will require one 7 digit alphanumeric code label on the cap of each tube.
Colorectal Cancer Family Registry - Coordinating Center Laboratory
University of Southern California, Department of Preventive Medicine
Colorectal Cancer Family Registry - Coordinating Center Laboratory
University of Southern California, Department of Preventive Medicine
Drafted October 10, 1998
Revised _______________
Drafted October 10, 1998
Revised Feb 13, 2004 AD
Biospecimen data records and report
1. Blood Log Sheet: The following will be written on a Blood Log Sheet before and
during processing:
a. study ID#
b. name of center
c. date drawn
d. time drawn (PST)
e. time of arrival (PST)
f. date of arrival
g. date processed
h. total blood volume in milliliters of EDTA tubes and ACD tubes
Vacutainer tubes contain the indicated amount of blood when filled 1/4” below the
stopper.
i. number of aliquots of plasma and lymphocytes
j. processing end time (PST)
Aliquots will be temporarily stored in a designated freezer location until a permanent
location is assigned. Once the permanent location is entered into the database, the tubes
will be moved.
2. Detailed report for each specimen
3. Inventory
Samples will be tracked using a high density laser scanner for barcode labels (once we
know, indicate the type). As previously stated, the CFR database in Access 97 will
provide the lab with more specific information about each sample. The CFR database will
be accessible to a limited number of staff, and will be stored on an computer in Room
5429 that is not on a network. A workgroup security system within Access 97 will be
activated, allowing only specified individuals with the password to edit the database.
These individuals are Anh Diep, Thanh Diep, and Owen Duffy. Immediately following
each data entry session, the database will be saved to the designated computer, a backup
ZIP disk that will be stored in the lab, and a backup ZIP disk that will be stored at an
offsite location. This backup procedure is specifically for the lab’s recordkeeping, and is
separate from the department’s usual archiving system. A periodical updates of the
database to Darthmouth via the internet or electronic transfer.
Colorectal Cancer Family Registry - Coordinating Center Laboratory
University of Southern California, Department of Preventive Medicine
Colorectal Cancer Family Registry - Coordinating Center Laboratory
University of Southern California, Department of Preventive Medicine
Drafted October 10, 1998
Revised Sept. 9, 1999 AD
Drafted October 10, 1998
Revised Feb 13, 2004 AD
Biospecimen database tables
Blood table
Blood subtable
Paraffin block table
Paraffin Slides table
Tissue table
Buccal smear table
DNA table
RNA table
Blood aliquot dispatch table
Paraffin block dispatch table
Paraffin slides dispatch table
Tissue dispatch table
DNA dispatch table
RNA dispatch table
Destination table
Colorectal Cancer Family Registry - Coordinating Center Laboratory
University of Southern California, Department of Preventive Medicine
Colorectal Cancer Family Registry - Coordinating Center Laboratory
University of Southern California, Department of Preventive Medicine
Drafted October 10, 1998
Revised _______________
Drafted October 10, 1998
Revised Feb 13, 2004 AD
QUALITY CONTROL
Colorectal Cancer Family Registry - Coordinating Center Laboratory
Drafted October 10, 1998
University of Southern California, Department of Preventive Medicine
Revised Feb 13, 2004 AD
1. Mix-up QC.
To evaluate the quality of the biospecimen handling throughout the entire process by
performing routine QC assays for randomly selected 10% of the specimens. As described
in DNA preparation protocol, DNA typing using microsatellite analyses with high
discriminating power allows us to compare the genotypes between aqueous DNA source
and bloodstain DNA source or any others from the same individual. It is the PCR setup of
less than 20 ng of DNA using D1S80 that is mapped at the distal of Chromosome 1 and
has the discriminating power of 95-98%. PCR product of 350 bp-1 kb size will be
electrophoresed on native agarose gel and then UV-visualize the genotypes.
Samples that are certified for future research will be confirmed to have identical
genotype patterns to that for other source of DNA from the same individual. Samples that
are identified, as mix-up will be excluded or hold for further resolution.
2. DNA Preparation QC
After every batch of DNA extraction, a series of assays are required to evaluate
the quality of this long term preserved genetic materials:
a. Determine DNA purity by taking its absorption spectra to obtain OD260/OD280
ratio.
b. Determine DNA integrity by running undigested DNA on a 1% agarose gel with a
standard of known concentration to observe for the intact band of DNA at high
molecular weight and for the smear at low molecular weight, which is the sign of
the degradation.
c. Optional to determine DNA digestibility with Hind III or Ecor I to ensure the
absence of trace organic solvents, salts, cellular proteins and proteinase K in the
final DNA solution, assuming that above a. and b. performance will be sufficient
to certify the DNA quality for long term storage.
Colorectal Cancer Family Registry - Coordinating Center Laboratory
University of Southern California, Department of Preventive Medicine
Colorectal Cancer Family Registry - Coordinating Center Laboratory
University of Southern California, Department of Preventive Medicine
Drafted October 10, 1998
Revised _______________
Drafted October 10, 1998
Revised Feb 13, 2004 AD
Summary of Routine QC for all specimens
(Adapted from BCSCFR)
Every six months, chose 5 individuals at random to test:
1) Cell lines
-
transform 5 cases
recover freezes from last 5 cases to test for re-growth, and to compare genotypes
vs. DNA made last time, and then refreeze
EBV cell line will be provided by Coriell and each lab will perform PCR tests for mycoplasma
contamination and Hoechst staining if possible - likely to be done in BNE at first
2) DNA
3) RNA]
-
isolate DNA from BP freeze from the same 5 test cases and matching Guthrie
spots (if not already done for other purposes) and from LCL to check:
OD 260/280
Agarose gel
Cut + uncut (high and low salt) Eco RI: Hind III
PCR [4 STRs and Y- chromosome PCR]
isolate RNA from the same 5 test cases from WBC freeze (if not already done)
and check:
OD 260/280 or Orcinol
Agarose gel (1.2%)
RT-PCR using AS promers (~ 1 kb) and PGK primers (247 bp)
Once a year, send DNA, RNA to another group to test.
At six month intervals:
.
.
.
.
Transform one aliquot of frozen white blood cells from 5 individual
Prepare DNA from whole blood from 5 individuals
Prepare DNA from blood spots from the same 5 individuals
Prepare RNA from one aliquot of frozen white blood cells from 5 individuals
Lymphoblastoid Cell Line:
. Viability
. Myoplasma contamination (PCR)1
DNA from whole blood:
. OD260/OD280
. Agarose gel electrophoresis
. Restriction digest with EcoRI and HindIII
. Microsatellite analysis with two markers; D10S526 and D22S4172
. Gender analysis if there is a problem
Colorectal Cancer Family Registry - Coordinating Center Laboratory
University of Southern California, Department of Preventive Medicine
Colorectal Cancer Family Registry - Coordinating Center Laboratory
University of Southern California, Department of Preventive Medicine
Drafted October 10, 1998
Revised _______________
Drafted October 10, 1998
Revised Feb 13, 2004 AD
DNA from Blood Spots:
. Microsatellite analysis with two markers; D10S526 and D22S4172
. Confirm that this pattern is identical to that for DNA from whole blood from the same
individual.
DNA from whole blood:
. OD260/OD280 or Orcinol
. Agarose gel (1- 2%) electrophoresis
. RT-PCR using 2 sets of primers: p53 (1248 bp), PGK (247 bp)3
See Detailed Protocol Section from Philadelphia
See addendum to Minutes of February 25, 1997 for sequences and PCR conditions
See addendum to Minutes of January 27, 1997 for sequences and PCR conditions
Colorectal Cancer Family Registry - Coordinating Center Laboratory
University of Southern California, Department of Preventive Medicine
Colorectal Cancer Family Registry - Coordinating Center Laboratory
University of Southern California, Department of Preventive Medicine
Drafted October 10, 1998
Revised _______________
Drafted October 10, 1998
Revised Feb 13, 2004 AD
General protocol for QC and QA
Sample processing
1. Adopt a barcode system to ensure tight specimen control and handling.
2. General laboratory biosafety and contamination control protocols have to be followed
(sterilization, for DNA/RNA/cell line, etc)
3. Ensure specimen viability (cells and cell lines ) by routine QC test.
4. Ensure specimen integrity by routine QC test and inter-center exchange plan.
During experiment
1. Again, strict laboratory contamination control has to be followed (sterilization, for
DNA/RNA/cell line, etc)
See lab setup protocol for details regarding biosafety, PCR, creating a ribonuclease-free
environment, etc.
2. All assays will include a “positive” control and a “negative” control.
3. For high volume experiments, no consecutive samples should be run by the same
technician.
4. If the study provides high yield specimens and has sufficient funding, all samples should
be run in duplicate.
5. If discrepancies are found from QC test, repeat experiments will be run by a different lab
technician in a blind manner.
6. If discrepancies are found from QC test, we can even have the collaborator repeat the
experiment in a blind manner.
7. Repeat experiments will be performed for all “positives” detected in a study when
“positives” (mutants) are rare.
8. Randomly pick from original materials to repeat some “positives” detected when the
“positives” rate is high.
9. All repeat QC test will work from redrawn blood, or QC source (blood spots), or stock
DNA in a blind manner (instead of previous amplified PCR products).
Data analysis
1. Data will be scored by multiple readers.
2. Each reader will independently score the data in a blind manner.
Data entry
1.
2.
3.


Records will be scored into database independently.
A second reader also will confirm the score independently.
Have strict editing protection by:
limited access of lab staff to database ( password permission ).
have a record of date and time editing monitored by computer software.
Colorectal Cancer Family Registry - Coordinating Center Laboratory
University of Southern California, Department of Preventive Medicine
Drafted October 10, 1998
Revised _______________
Colorectal Cancer Family Registry - Coordinating Center Laboratory
Drafted October 10, 1998
University of Southern California, Department of Preventive Medicine
Revised Feb 13, 2004 AD
Date:__________Int’ :____equipment used:
DNA Extraction Worksheet
From Buffy Coat
DNA quantitation by spectrophotometer
Record of sample ID, A260, A280 and A260/A280 from 1 ul of DNA solution. Calculate DNA concentration in ng/ul.
(OD260*dilution factor*coefficient for DNA where dilution factor is 100, and the coefficient for DNA is 50. )
Calculate total DNA yield and record working dilution preparation.
Tape on this the OD reading printouts*. Highlight samples that have A260/A280 not in 1.8 -2.0 range.
(These samples are to be re-extracted again.)
Comments:
__________________________________________________________________________________________
Colorectal Cancer Family Registry - Coordinating Center Laboratory
Uni versity of Southern California, Department of Preventive Medicine
Drafted Feb. 8, 1999
Revised Feb 11, 1999 AD
Colorectal Cancer Family Registry - Coordinating Center Laboratory
Drafted October 10, 1998
University of Southern California, Department of Preventive Medicine
Revised Feb 13, 2004 AD
DNA Extraction Worksheet
From Buffy Coat
Date:__________Int’:___
Examine DNA integrity and digestibility by gel electrophoresis
Make sure digestion sample list is correspond to extraction sample list.
# of samples
Reaction setup
1x, ul
DNA
50 ul
ddH2O
3.5 ul
10x buffer
6 ul
[DNA]
4 ng/ul
Enzyme
0.5 ul
DNA amount, ul
200 ng, 50 ul
Total digestion volume
60 ul
Enzyme
Hind III
from
Gibco BRL
Boost digestion
(if need)
[enzyme]
10 u/ ul
ddH2O
Enzyme amount, ul
1-5 u, 0.5 ul
10x buffer
buffer system
REact 2
50 mM Tris-HCl, pH 8.0
10 mM MgCl2
50 mM NaCl
Additional digestion V
Incubation T and duration
37C, 4 hrs
____x, ul
Enzyme
Gel loading prep
digestion mixture
18 ul
10x loading buffer
2 ul
Total loading volume
20 ul
Run cut and uncut DNA* with a standard of known concentration.
Highlight to repeat digestion of any samples that were either undigestible with restriction enzymes or samples that are
severely degraded.
* Make sure gel running sample list is correspond to extraction sample list.
Apparatus model
horizotal
Gel kind
Agarose
Gel %
1%
Photo of gel
Gel size
Gel Component
g/ml buffer
Comb size
Well capacity
20-25 ul
Buffer
0.6 x TBE
Direct Current
Time
:
to
:
Notes:
Comments:
__________________________________________________________________________________________
Colorectal Cancer Family Registry - Coordinating Center Laboratory
Uni versity of Southern California, Department of Preventive Medicine
Drafted Feb. 8, 1999
Revised Feb 11, 1999 AD
Colorectal Cancer Family Registry - Coordinating Center Laboratory
Drafted October 10, 1998
University of Southern California, Department of Preventive Medicine
Revised Feb 13, 2004 AD
DNA Extraction Worksheet
From Buffy Coat
Date:__________Int’’:
DNA Typing by VNTR analyses
* Make sure typing sample list is correspond to extraction sample list.
Marker Information
VNTR
Order
PCR setup
[stock]
[final]
1x, ul
1
ddH2O
—
—
7.4 ul
16-bp repeat unit
Locus
D1S80
2
10x buffer
—
1x
2 ul
Chromosomal localization
1p distal
3
dNTPs
2.5 mM
0.25 mM
2 ul
Allele size range
350 bp-1 kb
4
Primer 1
2 uM
4 pmol
2 ul
Heterozygosity
90% in Caucasians
5
Primer 2
2 uM
4 pmol
2 ul
Allelic frequency
29 alleles identified
435 possible genotypes
6
DNA polymerase
5 u/ ul
2u
0.4 ul
Discrimination power
95-98%
Primer code
7
XB20, XC01
8
AmplFLP D1S80 Allelic Ladder PERKIN ELMER N808-0064
27 amplified alleles
MgCl2
25 mM
1.5 mM
1.2 ul
Total Master Mix
—
—
17 ul
Template
10 ng/ ul
30 ng
Final volume
____x, ul
3 ul
20 ul
allele #14, 16-41
PCR Profile:
1 cycle @ 94C for 4 min. 35 cycle of 94C denaturation for
1 min, 65C annealing for 1 min and 72C elongation for 8 min. 1 final cycle of
72C elongation for 5 min. Hold @ 4C for further steps.
AmplFLP D1S80
Allelic Ladder, lab code
Run PCR product with AmplFLP D1S80 Allelic Ladder.
* Make sure gel running sample list is correspond to extraction sample list.
Apparatus model
horizontal
Gel kind
Agarose
Gel %
1.5%
Photo of gel
Gel size
Gel Component
g/ml buffer
Comb size
Well capacity
20-25 ul
Buffer
0.6x TBE
Loading buffer
ficoll dye
Loading volume
22.5 ul
Direct Current
Time
:
to
:
Notes:
 Load mixture of
5 ul /lane Allelic
Ladder + 2.5 10x dye
= 22.5 ul
+ 15 ddH2 O
Load 20 ul (-)
= 22.5 ul (-)
PCR control
PCR cont.
Comments:
__________________________________________________________________________________________
Haile’s Genetic Epidemiology Laboratory
Uni versity of Southern California, Department of Preventive Medicine
Drafted Feb. 8, 1999
Revised Jun 21, 2001 AD
Colorectal Cancer Family Registry - Coordinating Center Laboratory
Drafted October 10, 1998
University of Southern California, Department of Preventive Medicine
Revised Feb 13, 2004 AD
Mix-Up Quality Control for DNA
Blood spot or
other DNA source
Buffy coat
extract DNA
Is OD 260/280
ratio between
1.7-1.9?
No
perform
purification
Is undigested
DNA giving
intact band by
agarose gel
electrophoresis?
Yes
No
extract DNA
from another
aliquot or
from other
source
Is undigested
DNA giving
intact band by
agarose gel
electrophoresis?
No
extract DNA
No
Yes
Perform microsatellite analyses
Certify for
CFR
Repository
Is OD 260/280
ratio between
1.7-1.9?
Yes
Perform microsatellite analyses
Is it feasible to obtain PCR product?
Is microsatellite pattern identical with other DNA source?
No
Ship DNA as
instructed
Request
Submission
Colorectal Cancer Family Registry - Coordinating Center Laboratory
Drafted October 10, 1998
University of Southern California, Department of Preventive Medicine
Revised Feb 13, 2004 AD