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.
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.
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
excitation scanning cDNA clones (probes) laser 2 laser 1 PCR product amplification purification emission printing mRNA target) overlay images and normalise 0.1nl/spot Hybridise target to microarray microarray analysis
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
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.
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.
M-Guide: Build your own arrayer • M-Guide • Array Maker Documentation • Printing Microarrays
Printing Microarrays • Print Head • Plate Handling • XYZ positioning • Repeatability & Accuracy • Resolution • Environmental Control • Humidity • Dust • Instrument Control • Sample Tracking Software
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
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
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
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.
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
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.
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.
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.
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
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)
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.
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
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)
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
Hybridization • Humidity • Temperature • Formamide (Lowers the Tm) 3XSSC HYB CHAMBER ARRAY LIFTERSLIP SLIDE LABEL SLIDE LABEL
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.
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.
Micrograph of a portion of hybridization probe from a yeast mciroarray (after hybridization).
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)
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.
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
Biological Question Data Analysis & Modeling Samplepreparation Microarray Life Cycle MicroarrayDetection Microarray Reaction Taken from Schena & Davis