cDNA microarrays on glass
slides
An overview of the Brown-De Risi- Iyer technology,
based on
—the 2000 CSH Microarray Course notes, Nature
Genetics Supp, Jan 1999,
—two books edited by M Schena: DNA Microarrays, A
Practical Approach, OUP 1999, and Microarray Biochip
Technology, Eaton Publishing, 2000,
—DNA Arrays or Analysis of Gene Expression by
M. Eisen and P. Brown, and
—the experiences of my colleagues.
Department of Statistics, University of California, Berkeley, and
Division of Genetics and Bioinformatics, The Walter and Eliza Hall Institute of Medical Research,
History
cDNA microarrays have evolved from Southern blots,
with clone libraries gridded out on nylon membrane
filters being an important and still widely used
intermediate. Things took off with the introduction of
non-porous solid supports, such as glass - these
permitted miniaturization - and fluorescence based
detection. Currently, about 20,000 cDNAs can be
spotted onto a microscope slide. The other, Affymetrix
technology can produce arrays of 100,000
oligonucleotides on a silicon chip. I will not discuss
these further.
Department of Statistics, University of California, Berkeley, and
Division of Genetics and Bioinformatics, The Walter and Eliza Hall Institute of Medical Research,
THE PROCESS
Building the Chip:
MASSIVE PCR
PCR PURIFICATION
and PREPARATION
PREPARING SLI DES
PRINTING
Preparing RNA:
CELL CULTURE
AND HARVEST
Hybing the Chip:
POST PROCESSING
ARRAY HYBRIDIZATION
RNA ISOLATION
DATA ANALYSIS
cDNA PRODUCTION
PROBE LABELING
Department of Statistics, University of California, Berkeley, and
Division of Genetics and Bioinformatics, The Walter and Eliza Hall Institute of Medical Research,
excitation
cDNA clones
(probes)
laser 2
PCR product amplification
purification
printing
scanning
laser 1
emission
mRNA target)
overlay images and normalise
0.1nl/spot
microarray
Hybridise target
to microarray
analysis
Department of Statistics, University of California, Berkeley, and
Division of Genetics and Bioinformatics, The Walter and Eliza Hall Institute of Medical Research,
Department of Statistics, University of California, Berkeley, and
Division of Genetics and Bioinformatics, The Walter and Eliza Hall Institute of Medical Research,
Building the Chip:
MASSIVE PCR
Full yeast genome
= 6,500 reactions
PREPARING SLI DES
Polylysine coating for adhering
PCR products to glass slides
PCR PURIFICATION
and PREPARATION
IPA precipitation + EtOH
washes + 384-well format
PRINTING
The arrayer: high precision spotting device
capable of printing 10,000 products in 14 hrs,
with a plate change every 25 mins
POST PROCESSING
Chemically converting the positive
polylysine surface to prevent nonspecific hybridization
Department of Statistics, University of California, Berkeley, and
Division of Genetics and Bioinformatics, The Walter and Eliza Hall Institute of Medical Research,
Preparing RNA:
CELL CULTURE
AND HARVEST
Designing experiments to profile conditions/perturbations/
mutations and carefully controlled growth conditions
RNA ISOLATION
RNA yield and purity are determined by system. PolyA isolation is preferable
but total RNA is useable. Two RNA samples are hybridized/chip.
cDNA PRODUCTION
Single strand synthesis or amplification of RNA can be performed.
cDNA production includes incorporation of Aminoallyl-dUTP.
Department of Statistics, University of California, Berkeley, and
Division of Genetics and Bioinformatics, The Walter and Eliza Hall Institute of Medical Research,
Hybing the Chip:
ARRAY HYBRIDIZATION
Cy3 and Cy5 RNA samples are simultaneously
hybridized to chip. Hybs are performed for 5-12 hours
and then chips are washed.
DATA ANALYSIS
PROBE LABELING
Two RNA samples are labelled with Cy3 or
Cy5 monofunctional dyes via a chemical
coupling to AA-dUTP. Samples are purified
using a PCR cleanup kit.
Ratio measurements are determined via
quantification of 532 nm and 635 nm
emission values. Data are uploaded to the
appropriate database where statistical and
other analyses can then be performed.
Department of Statistics, University of California, Berkeley, and
Division of Genetics and Bioinformatics, The Walter and Eliza Hall Institute of Medical Research,
M-Guide: Build your own
arrayer
• M-Guide
• Array Maker Documentation
• Printing Microarrays
Department of Statistics, University of California, Berkeley, and
Division of Genetics and Bioinformatics, The Walter and Eliza Hall Institute of Medical Research,
Printing Microarrays
• Print Head
• Plate Handling
• XYZ positioning
– Repeatability & Accuracy
– Resolution
• Environmental Control
– Humidity
– Dust
• Instrument Control
• Sample Tracking Software
Department of Statistics, University of California, Berkeley, and
Division of Genetics and Bioinformatics, The Walter and Eliza Hall Institute of Medical Research,
Ngai Lab arrayer , UC Berkeley
Department of Statistics, University of California, Berkeley, and
Division of Genetics and Bioinformatics, The Walter and Eliza Hall Institute of Medical Research,
Microarray Gridder
Department of Statistics, University of California, Berkeley, and
Division of Genetics and Bioinformatics, The Walter and Eliza Hall Institute of Medical Research,
Slide Preparation: Home Grown
Protocol for preparing poly-L-Lysine slides
for Microarrays
1. Wash 180 slides completely
2. Prepare poly-lysine solution
3. Pour solution over slide
4. Rinse, spin dry and store slides
5. Use slides no less than 2 and no
more than 4-6 months later
Department of Statistics, University of California, Berkeley, and
Division of Genetics and Bioinformatics, The Walter and Eliza Hall Institute of Medical Research,
Department of Statistics, University of California, Berkeley, and
Division of Genetics and Bioinformatics, The Walter and Eliza Hall Institute of Medical Research,
Product Amplification and preparation: What to Print?
Protocol for Amplifying Products
to Print on Array
All PCR reactions in 96-well format, 100 ml reaction volume
Perform PCR reactions in a Tetrad Machine
Reactions are assayed on 96 well agarose gel
Need multi-channel pipetting system
Also desirable to have Multimek 96-well pipetting robot
Department of Statistics, University of California, Berkeley, and
Division of Genetics and Bioinformatics, The Walter and Eliza Hall Institute of Medical Research,
MJ Tetrad PCR machine
Department of Statistics, University of California, Berkeley, and
Division of Genetics and Bioinformatics, The Walter and Eliza Hall Institute of Medical Research,
Protocol for preparation of Plasmid
DNA from Bacterial Clones Containing
Mammalian DNA
1. Inoculate a deep 96-well plate filled with IB (+
antibiotic marker) with a small amount of
bacterial culture. Incubate with shaking at
37˚C
2. Spin down the cultures and follow the
manufacturer’s protocol for the QIAprep
3. Use 1-5 ul of eluted plasmid DNA as PCR
template
Department of Statistics, University of California, Berkeley, and
Division of Genetics and Bioinformatics, The Walter and Eliza Hall Institute of Medical Research,
Protocol for precipitation and 384
Well Arraying of PCR products
1. After running reactions on 1% agarose gel and documenting
results, add sodium acetate, pH 5.5 and 110 ul room temp
isopropanol
2. Transfer reactions to U-bottom plates,.. tape plates together.
3. Spin plates at 4.500 rpm for 2 hours
4. Carefully aspirate solution
5. Add 100ul 70% EtOH. Spin plates for another hour at 4,500
6. Aspirate again and let air dry or dry in a 96 well speed-vac
7. Allow PCR products to resuspend in 20ul of H2O for at least 18
hours
8. Transfer products to 384 -well printing plates
9. Dry plates down in speed-vac and resuspend products in 3X SSC
10. Let plates resuspend overnight before printing.
Department of Statistics, University of California, Berkeley, and
Division of Genetics and Bioinformatics, The Walter and Eliza Hall Institute of Medical Research,
Department of Statistics, University of California, Berkeley, and
Division of Genetics and Bioinformatics, The Walter and Eliza Hall Institute of Medical Research,
Printing Approaches
Non - Contact
• Piezoelectric dispenser
• Syringe-solenoid ink-jet dispenser
Contact (using rigid pin tools, similar to filter
array)
• Tweezer
• Split pin
• Micro spotting pin
Department of Statistics, University of California, Berkeley, and
Division of Genetics and Bioinformatics, The Walter and Eliza Hall Institute of Medical Research,
Micro Spotting
pin
Department of Statistics, University of California, Berkeley, and
Division of Genetics and Bioinformatics, The Walter and Eliza Hall Institute of Medical Research,
Department of Statistics, University of California, Berkeley, and
Division of Genetics and Bioinformatics, The Walter and Eliza Hall Institute of Medical Research,
Practical Problems
— Surface chemistry: uneven surface may lead to high
background.
— Dipping the pin into large volume -> pre-printing to
drain off excess sample.
— Spot variation can be due to mechanical difference
between pins. Pins could be clogged during the
printing process.
— Spot size and density depends on surface and
solution properties.
— Pins need good washing between samples to prevent
sample carryover.
Department of Statistics, University of California, Berkeley, and
Division of Genetics and Bioinformatics, The Walter and Eliza Hall Institute of Medical Research,
Post Processing Arrays
Protocol for Post Processing Microarrays
Hydration/Heat Fixing
1. Pick out about 20-30 slides to be processed.
2. Determine the correct orientation of slide, and if necessary, etch
label on lower left corner of array side
3. On back of slide, etch two lines above and below center of array to
designate array area after processing
4. Pour 100 ml 1X SSC into hydration tray and warm on slide warmer
at medium setting
5. Set slide array side down and observe spots until proper hydration
is achieved.
6. Upon reaching proper hydration, immediately snap dry slide
7. Place slides in rack.
Department of Statistics, University of California, Berkeley, and
Division of Genetics and Bioinformatics, The Walter and Eliza Hall Institute of Medical Research,
Surface blocking
1. Store succinic anhydride in vacuum dessicator until ready
for use.
2. Measure 335 ml 1-methly-2-pyrrolidinone into designated
clean dry slide dish with stir bar
3. Dissolve 5.5 g succinic anhydride completely
4. IMMEDIATELY after succinic anhydride dissolves, mix in 15
ml 1M NaBorate pH 8.0 and submerge slides in solution.
Shake evenly under level of solution.
5.Soak slides in solution on shaker for 15’
6. Before 15’ incubation is done, reduce heat on boiling water
so temp is approx 95C and no more bubbles are present.
Drain excess blocking solution off slides and transfer slide
rack to boiling water and incubate for 1’30”
7. Transfer rack to dish of 95% EtOH and plunge 5X. Spin
down on tabletop.
8. Arrays may be used immediately or stored for future use.
Department of Statistics, University of California, Berkeley, and
Division of Genetics and Bioinformatics, The Walter and Eliza Hall Institute of Medical Research,
Isolating Nucleic Acid:
“RNA, Membranes, and Tumors”
Protocol for Total RNA isolation in S. Cerevisae
Modified FastTrack Protocol for Yeast Poly-A RNA
Isolation
Protocol for Poly-A Isolations
Revised Protocol for FastTrack mRNA extraction from
Human Cells
Tumor mRNA isolation
Gradient-based membrane-bound Polysome Protocol
Protocol for Immunoprecipitation of Chromatin from
Fixed Yeast Beadbeater Method
Department of Statistics, University of California, Berkeley, and
Division of Genetics and Bioinformatics, The Walter and Eliza Hall Institute of Medical Research,
Department of Statistics, University of California, Berkeley, and
Division of Genetics and Bioinformatics, The Walter and Eliza Hall Institute of Medical Research,
Protocol for Total RNA Isolation in
S. Cerevisae
1. Spin down cells (about 250ml at OD600=0.5). Dump
supernatant.
2. Resuspend in 12 ml of AE Buffer. Transfer to Oak
Ridge phenol resistant centrifuge tubes.
3. Add 800 ul 25% SDS, 12 ml acid phenol. Mix well.
4. Incubate 10’ at 65 ˚C, vortexing every minute.
5. Incubate 5’ on ice.
6. Spin down 15 minutes at 10,000 rpm in SS34 rotor
7. Dump supernatant into pre-spun 50 ml PhaseLock
tube.Add 15 ml chloroform and shake to mix (…ctd)
Department of Statistics, University of California, Berkeley, and
Division of Genetics and Bioinformatics, The Walter and Eliza Hall Institute of Medical Research,
8. Spin down 10’ at 3,000 rpm in table-top centrifuge
9. Dump supernatant into new oak Ridge tube
10. Add 1/10 volume 3M NaAcetate and equal volume of room
temperature isopropanol
11. Spin down 35’-40’ at 12,000 rpm in SS34
12. Wash with 70% EtOH, resuspending pellet, spin again 20’ at
12,000 rpm
13. Dump off EtOH. Dry pellet in vacuum oven briefly
14. Resuspend in 500ul water
15. Quantitate via spec and run 1ug on 1% agarose gel
16. Store total RNA in -80˚C
Protocol for Poly-A Isolations more complex: 18 steps.
Department of Statistics, University of California, Berkeley, and
Division of Genetics and Bioinformatics, The Walter and Eliza Hall Institute of Medical Research,
Labelling Nucleic Acid
Protocol for Reverse transcription and Amino-allyl
Coupling of RNA
Preparation of Fluorescent cDNA Probe from Human
mRNA (alternate protocol)
Modified Eberwine (“ANTISENSE”) RNA Amplification
Protocol
Protocol for labeling Genomic DNA for Microarrays Version 1
Genomic DNA Labeling Protocol
Round A/B DNA Ampification Protocol
Department of Statistics, University of California, Berkeley, and
Division of Genetics and Bioinformatics, The Walter and Eliza Hall Institute of Medical Research,
Preparation of Fluorescent cDNA
Probe from Human mRNA
(alternate protocol)
1. To anneal primer, mix 2 ug of mRNA with 2 ug of a regular or
anchored (5’-TTT TTT TTT TTT TTT TTT TT VN-3’) oligo-dT primer
in a total volume of 15 ul (x 2)
2. Heat to 70 ˚C for 10 min and cool on ice
3. Add 15 ul of reaction mixture each to Cy3 and Cy5 reactions (5X
first strand buffer, 0.1M DTT, unlabeled dNTPs, Cy3 or Cy5,
Superscript II
4. 5X first strand buffer: 250 mM Tris-HCl, 375 KCl, 15mM MgCl2
5. Incubate at 42 ˚C for 1.5-2 hrs
6. Degrade RNA by addition of 15ul of 0.1N NaOH, and incubate at 70
˚C ……(ctd)
Department of Statistics, University of California, Berkeley, and
Division of Genetics and Bioinformatics, The Walter and Eliza Hall Institute of Medical Research,
7. Neutralize by addition of 15 ul of 0.1N HCl, and bring the volume to 500 ul with TE
8. Add 20 ug of Cot1 hman DNA
9. Purify probe by centrifuging in a Centricon micro-concentrator
-------------------------------------------------------------------------------------10. Combine the separate concentrated probes (Cy3 and Cy5) into a fresh Centricon,
bring to a volume of 500 ul with TE and concentrate again
11. Add 1 ul of 10ug/ul polyA RNA and 1 ul of 10ug/ul tRNA
12. Adjust volume to 9.5 ul with distilled water
13. For final probe preparation add 2.1 ul 20XSSC and 0.35 ul 10% SDS. Final probe
volume can be adjusted to between 12 ul and 15 ul.
14. Denature probe by heating for 2 min at 100 ˚C, and incubate at 37 ˚C for 20-30 min
15. Place on array under a glass cover slip
16. Hybridize at 65 ˚C for 14 to 18 hours in a custom slide chamber with humidity
maintained by a small reservoir of 3XSSC
17. Wash arrays by submersion and agitation for 2-5 min in 2X SSC with 0.1%SDS
followed by 1X SSC and 0.1X SSC
18. Spin dry by centrifugation for 2c min on a slide rack in a tabletop centrifuge at 650
rpm for 2min
Department of Statistics, University of California, Berkeley, and
Division of Genetics and Bioinformatics, The Walter and Eliza Hall Institute of Medical Research,
Hybridization
• Humidity
• Temperature
• Formamide
(Lowers the Tm)
3XSSC
HYB CHAMBER
ARRAY
LIFTERSLIP
SLIDE
LABEL
SLIDE LABEL
Department of Statistics, University of California, Berkeley, and
Division of Genetics and Bioinformatics, The Walter and Eliza Hall Institute of Medical Research,
Hybridization Chamber
Department of Statistics, University of California, Berkeley, and
Division of Genetics and Bioinformatics, The Walter and Eliza Hall Institute of Medical Research,
Protocol for Array Hybridization
1. Prepare probe as described at the end of the labeling
protocol
2, Set slide in hybridisation chamber
3. Clean a lifterslip with EtOH and Kimwipes. Place slip on
array using either fingers or forceps
4. Boil probe for 2 min at 100 ˚C. Let cool 5-10 min at room
temp.
5. Slowly inject the probe under one corner of the cover slip
until the array surface is covered. Continue to apply
remaining probe at the other corners.
6. Tightly screw down chamber lid and carefully place
chamber in a 63˚C water bath,
7. Allow hybridisation to run at least 5 hours but not more than
16 hours.
Department of Statistics, University of California, Berkeley, and
Division of Genetics and Bioinformatics, The Walter and Eliza Hall Institute of Medical Research,
Array Washing Protocol
Details of 7-stage washing protocol skipped but it is
a very important step.
…………...
8. Try to scan array within hours of washing as the
Cy dyes are unstable and will degrade
differentially.
Department of Statistics, University of California, Berkeley, and
Division of Genetics and Bioinformatics, The Walter and Eliza Hall Institute of Medical Research,
Micrograph of a portion of hybridization probe from
a yeast mciroarray (after hybridization).
Department of Statistics, University of California, Berkeley, and
Division of Genetics and Bioinformatics, The Walter and Eliza Hall Institute of Medical Research,
GenePix 4000a Microarray
Scanner Protocol
1. Turn on scanner.
2. Slide scanner door open. Insert chip hyp side down and clip chip holder
easily around the slide
3 Set PMTs to 600 in both 635nm (Cy3) and 532 (Cy5) channels.
4. Perform low resolution “PREVIEW SCAN” to determine location of spots
and initial hyb intensities
5. Once scan location determined, draw a “SCAN AREA” marquis around the
array
6. Perform quick visual inspection of hyb and make initial adjustments to
PMTs
7. For gene expression hybs, raise or lower the red and green PMTs to
achieve color balance
8. Before you perform your data scan, change “LINES TO AVERAGE” to 2.
9. Perform a high-resolution “DATA-SCAN”……(ctd)
Department of Statistics, University of California, Berkeley, and
Division of Genetics and Bioinformatics, The Walter and Eliza Hall Institute of Medical Research,
GenePix 4000a Microarray
Scanner Protocol, ctd
10. Observe the histograms and make adjustments to PMTs.
11. Once the PMT level has been set so that the Intensity Ratio is
near 1.00 perform a “DATA SCAN” over “SCAN AREA” and save
the results.
12. To save your image, select “SAVE IMAGES”.
13. Save as type=Multi-image TIFF files.
14. Once scanned and saved, you are ready to assign spot identities
and calculate results.
Note: For us, normalization is performed later during data analysis.
Department of Statistics, University of California, Berkeley, and
Division of Genetics and Bioinformatics, The Walter and Eliza Hall Institute of Medical Research,
Department of Statistics, University of California, Berkeley, and
Division of Genetics and Bioinformatics, The Walter and Eliza Hall Institute of Medical Research,
Summary of analysis possibilities
Determine genes which are differentially expressed (this task
can take many forms depending on replication, etc)
Connect differentially expressed genes to sequence
databases and perhaps carry out further analyses, e.g.
searching for common upstream motifs
Overlay differentially expressed genes on pathway diagrams
Relate expression levels to other information on cells, e.g.
known tumour types
Define subclasses (clusters) in sets of samples (e.g. tumours)
Identify temporal or spatial trends in gene expression
Seek roles for genes on the basis of patterns of co-expression
……..much more
Many challenges: transcriptional regulation involves
redundancy, feedback, amplification, .. non-linearity
Department of Statistics, University of California, Berkeley, and
Division of Genetics and Bioinformatics, The Walter and Eliza Hall Institute of Medical Research,
Biological
Question
Data Analysis
& Modeling
Microarray
Life Cycle
Sample
preparation
Microarray
Detection
Taken from Schena & Davis
Microarray
Reaction
Department of Statistics, University of California, Berkeley, and
Division of Genetics and Bioinformatics, The Walter and Eliza Hall Institute of Medical Research,