cDNA microarrays on glass slides

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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.

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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.

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THE PROCESS
Building the Chip
PCR PURIFICATION and PREPARATION
MASSIVE PCR
PREPARING SLIDES
PRINTING
Preparing RNA
Hybing the Chip
CELL CULTURE AND HARVEST
POST PROCESSING
ARRAY HYBRIDIZATION
RNA ISOLATION
DATA ANALYSIS
PROBE LABELING
cDNA PRODUCTION
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excitation
scanning
cDNA clones (probes)
laser 1
laser 2
PCR product amplification purification
emission
printing
mRNA target)
overlay images and normalise
0.1nl/spot
Hybridise target to microarray
microarray
analysis
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Building the Chip
PCR PURIFICATION and PREPARATION
MASSIVE PCR
Full yeast genome 6,500 reactions
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
PREPARING SLIDES
Polylysine coating for adhering PCR products to
glass slides
POST PROCESSING
Chemically converting the positive polylysine
surface to prevent non-specific hybridization
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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.
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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
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.
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.
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M-Guide Build your own arrayer

• M-Guide
• Array Maker Documentation
• Printing Microarrays

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Printing Microarrays

• Print Head
• Plate Handling
• XYZ positioning
• Repeatability Accuracy
• Resolution
• Environmental Control
• Humidity
• Dust
• Instrument Control
• Sample Tracking Software

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Ngai Lab arrayer , UC Berkeley
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Microarray Gridder
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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

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Protocol for Amplifying Products to Print on Array
Product Amplification and preparation What to
Print?

• 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

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MJ Tetrad PCR machine
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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 37C
• 2. Spin down the cultures and follow the
  manufacturers protocol for the QIAprep
• 3. Use 1-5 ul of eluted plasmid DNA as PCR
  template

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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.

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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

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Micro Spotting pin
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Practical Problems

• Surface chemistry uneven surface may lead to
  high background.
• Dipping the pin into large volume -gt 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.

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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.

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• 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 130
• 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.

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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

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Protocol for Total RNA Isolation in S.
Cerevisae

• 1. Spin down cells (about 250ml at OD6000.5).
  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)

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• 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 -80C
• Protocol for Poly-A Isolations more complex 18
  steps.

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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

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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)

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• 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 0.1SDS 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

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Hybridization

• Humidity
• Temperature
• Formamide
• (Lowers the Tm)

3XSSC
HYB CHAMBER
ARRAY
LIFTERSLIP
SLIDE
LABEL
SLIDE LABEL
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Hybridization Chamber
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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 63C water bath,
• 7. Allow hybridisation to run at least 5 hours
  but not more than 16 hours.

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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.

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Micrograph of a portion of hybridization probe
from a yeast mciroarray (after hybridization).
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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)

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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 typeMulti-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.

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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

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Biological Question
Data Analysis Modeling
Sample preparation
Microarray Life Cycle
MicroarrayDetection
Microarray Reaction
Taken from Schena Davis