Affymetrix Array Protocol
(using Staphylococcus Aureus)
I.
RNA extraction
A. Take cells (stored in either Acetone:EtOH (1:1) or RNAlater) from the
-20°C freezer
* I usually have 4ml aliquots of cells in 4ml of freezing solution
(typically Acetone:EtOH) in 15ml conicles.
B. Centrifuge @ 3,250 rpm, 4°C, for 10 – 15 minutes to pellet cells. Dump
supernatant.
C. Resuspend cells in 4ml (or same original volume) Low Salt TES (150mM
NaCl, 78mM EDTA, 100mM Tris pH 7.5). Vortex to make sure that cells
are fully resuspended.
D. Centrifuge @ 3,250 rpm, 4°C, for 10 – 15 minutes to pellet cells. Dump
supernatant.
E. Resuspend cells in 4ml Low Salt TES and vortex, making sure cells are
fully resuspended.
F. Centrifuge @ 3,250 rpm, 4°C, for 10 – 15 minutes to pellet cells. Dump
supernatant.
G. For 4ml of cells, resuspend the pellets in 140µl each High Salt TES (2.5 M
NaCl, 78mM EDTA, 100mM Tris pH 7.5). Transfer to Falcon tubes for
more surface area.
H. Add 60µl Lysotaphin to each tube and incubate in 37°C water bath for 30
minutes or until the cells look good and goopy.
I. Remove cells from water bath and continue isolation of RNA using Qiagen
RNeasy mini kit.
1. Aliquot cells into 1.5ml eppendorf tube - 100µl/tube.
2. Add 350µl Buffer RLT to each tube.
**Initially I had been adding ß-ME to the RLT buffer, but after
reading where several labs were not adding it, I have no longer
been adding the ß-ME. My results have so far been the same.
3. Vortex to mix.
4. Add 250µl of 95-100% Ethanol to each tube and mix by pipetting up
and down.
5. Apply entire volume to a Qiaquick column then centrifuge at 10,000
rpm for 15 seconds.
6. Discard flow-through.
7. Apply 700µl Buffer RW1 to each column and centrifuge at 10,000
rpm for 15 seconds. Discard flow-through.
8. Put columns into new collection tubes provided with the kit. Apply
500µl Buffer RPE to each column and centrifuge for 15 seconds.
Discard flow-through.
9. Apply another 500µl Buffer RPE to each column and again, centrifuge
for 15 seconds. Discard flow-through.
10. Centrifuge at 10,000 rpm for an additional 10 minutes to ensure that
the membrane is completely dry.
11. Elute with 40µl of Nuclease free water and spec at 260/280.
II.
DNase Digestion
A. 20µg of total RNA are digested with RQ DNase per tube. I usually set up
a few reactions in order to get enough for several cDNA synthesis
reactions.
B. Up to 40µl of RNA can be used. Add 5µl of RQ DNase buffer and 5µl of
RQ1 Dnase.
C. Incubate the reactions in a 37°C water bath for 1 hour.
D. Remove from the water bath and add 50µl of nuclease free water to each
tube, bringing the volume up to 100µl.
E. Using the Qiagen RNeasy Purification kit, add 350µl of Buffer RLT
(without ß-ME) and vortex to mix.
F. Add 250µl of 95-100% ethanol to each sample and mix by pipetting up
and down. Apply the entire amount to a qiagen column.
G. Centrifuge at 10,000 rpm for 15 sec. Discard flow-through.
H. Transfer columns to new collection tubes and apply 500µl buffer RPE to
each column. Centrifuge for 15 secs and discard flow-through.
I. Apply another 500µl buffer RPE to each column. Centrifuge for 15
seconds and discard flow-through.
J. Centrifuge for an additional 10 minutes to ensure that the membrane is
dry.
K. Transfer columns to 1.5ml tubes provided with the kit. Elute two times,
each time using 30µl of nuclease free water. Combine like samples and
spec at 260/280.
III.
DNA contamination PCR
A. Combine the following in 0.6 ml tubes:
400ng of RNA template
1µl of forward primer and reverse primer each (ex: rplO and sarB)
bring the volume to 20µl with sterile water
B. Make a Master Mix: (per reaction)
10x buffer – 2.5µl
MgCl2 - 1.0µl
dNTPs – 0.5µl
Taq (1:10) – 1.0µl
C. Add 5µl of the Master Mix to each sample.
D. Cycle under the following conditions:
95ºC – 4 min
95ºC – 1 min----|
55ºC – 1 min | 29 cycles
72ºC – 2 min----|
72ºC – 10 min
4ºC – hold
E. After cycling is complete, load 5µl of each product on a 1% agarose gel.
Product = DNA contamination. (I always set up a positive chromosomal
sample and a negative sample with each set of reactions).
IV.
cDNA Synthesis
A. Put 10µg of RNA into a 1.6ml tube and dry down to 25µl.
B. Combine the following in a 96-well plate:
RNA (10µg)
Random primers (@250ng/µl)
Spike (poly-A RNA control)
25µl
3µl
2µl
** The spike control is diluted as follows BEFORE adding to the above
mixture. First, make a 1:20 dilution by adding 2µl of poly-A RNA
control to 38µl of dilution buffer. Then, make a 1:13 dilution by adding
2µl of the 1:20 dilution to 24µl of the dilution buffer. The 1:20 dilution
can be stored at -20°C and freeze-thawed up to 8 times before going bad.
C. Cycle reactions in PE 9700 thermalcycler:
70°C 10min
25°C 10min
4°C
hold
D. Prepare the following reaction mix and add to the wells while in the
thermalcyler at 4°C: (per reaction)
5 x First Strand Buffer
100mM dTT
10mM dNTPs
RNAse Inhibitor
Superscript II
12µl
6µl
3µl
1.5µl
7.5µl
E. Incubate in PE 9700:
25°C
37°C
42°C
70°C
4°C
10min
60min
60min
10min
hold
F. Can either proceed directly to next step or let the reactions stay in the
thermalcyler overnight.
G. Transfer the reactions to 1.5ml tubes. Add 20µl of 1N NaOH to each
reaction and mix. Incubate @ 65ºC for 30 minutes.
H. Add 20µl of 1N HCl for the titration.
I. Clean reactions with Qiagen PCR cleanup kit.
1. Add 500µl of Buffer PB to each reaction then apply the entire
amount to a Qiagen column.
2. Centrifuge @ 13,000 rpm for 60 seconds.
3. Dump the flow-through and put the column back into the
collection tube.
4. Wash by adding 750µl of Buffer PE to each column and centrifuge
for 1 minute.
5. Dump the flow-through and return column to the collection tube.
6. Centrifuge for an additional 10 minutes to ensure dryness of the
column.
7. Elute by adding 30µl of Buffer EB to the column and centrifuging
for 1 minute. Repeat the elution with another 30µl of Buffer EB.
8. Combine like reactions and spec at 260/280.
V.
cDNA Fragmentation
A. Once you have collected 3µg of cDNA you can proceed with the cDNA
fragmentation
B. Prepare the following reaction mix on ice:
10x One Phor-All Buffer
cDNA
DNase I mix
5µl
3µg (up to 42µl)
3µl
** 1x Buffer
270µl sterile water
30µl 10x One Phor-All Buffer
**DNase I mix
1.5µl Roche DNase
248.5µl 1x Buffer
C. Add the DNase I mix last, mix and immediately begin incubation.
D. Incubate the reactions at 37°C for EXACTLY 10 minutes, moving
immediately to 98 - 100°C for 10min.
E. For quality control, pre- and post-fragmented cDNA samples are analyzed
by agarose gel electrophoresis.
F. Place samples on ice while preparing a 2% agarose gel containing GelStar
Nucleic Acid Gel Stain at a dilution of 1:10000.
G. Analyze 5µl of fragmented cDNA and 500ng of unfragmented cDNA as a
control. Use 2.5µl of Ready-load 100bp DNA Ladder as marker for size
determination. The desired fragmentation result should yield a majority of
the DNA fragments within a distribution of 50-200 bases.
VI.
Terminal Labeling
A. Prepare the following reaction on ice using the Enzo BioArray Terminal
Labeling Kit:
5x Reaction Buffer
10x CoCl2
Bioting-ddUTP
Terminal Deoxynucleotide
Transferase
Fragmentation Product
12µl
6µl
1µl
2µl
40µl
B. Incubate the reaction at 37ºC for 60 minutes.
C. Add 1.5µl of 0.5M EDTA pH 8.0 to stop the reaction.
D. Store the labeled products at -20°C until ready to ship to University of
IOWA.
VII.
Shipping
Garry Hauser (or Jessica Linton)
University of Iowa
DNA Facility
321 EMRB
Iowa City, IA 52242
Ph (319) 335-7928
VIII. Reagents and Vendors
A.
B.
C.
D.
E.
F.
G.
H.
I.
J.
K.
L.
M.
N.
Qiagen RNeasy Mini Kit – Qiagen – Cat# 74104
RQ1 DNase – Promega – Cat# M6101
Taq – Roche – Cat# 11 146 173 001
Random primers – Invitrogen – Cat# 48190-011
Affymetrix GeneChip Eukaryotic Poly-A RNA Control – Affymetrix –
Cat# 900433
dNTPs (100mM dATP, dCTP, dGTP, dTTP) – Amersham Pharmacia
Biotech (GE HealthCare) – Cat# 27-2035-01
RNase Inhibitor – Ambion – Cat# 2684
Superscript II – Invitrogen – Cat# 18064-014
QIAquick PCR Purification Kit – Qiagen – Cat# 28104
10x One Phor-All Buffer – Amersham Pharmacia Biotech (GE
HealthCare) – Cat# 27-0901-02
DNase – Roche – Cat# 10 776 785 001
GelStar Nucleic Acid Gel Stain – Cambrex – Cat# 50535
Ready-load 100bp DNA Ladder – Invitrogen – Cat# 10380-012
Enzo BioArray Terminal Labeling Kit – Enzo LifeSciences – Cat#
42630