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.
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: MASSIVE PCR PCR PURIFICATION and PREPARATION PREPARING SLIDES PRINTING Preparing RNA: CELL CULTURE AND HARVEST RNA ISOLATION cDNA PRODUCTION Hybing the Chip : POST PROCESSING ARRAY HYBRIDIZATION PROBE LABELING DATA ANALYSIS
MASSIVE PCR PCR PURIFICATION and PREPARATION PREPARING SLIDES PRINTING Building the Chip: Full yeast genome = 6,500 reactions IPA precipitation +EtOH washes + 384-well format The arrayer: high precision spotting device capable of printing 10,000 products in 14 hrs, with a plate change every 25 mins 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 RNA ISOLATION cDNA PRODUCTION Designing experiments to profile conditions/perturbations/ mutations and carefully controlled growth conditions RNA yield and purity are determined by system. PolyA isolation is preferable but total RNA is useable. Two RNA samples are hybridized/chip. Single strand synthesis or amplification of RNA can be performed. cDNA production includes incorporation of Aminoallyl-dUTP.
Hybing the Chip : ARRAY HYBRIDIZATION PROBE LABELING DATA ANALYSIS Cy3 and Cy5 RNA samples are simultaneously hybridized to chip. Hybs are performed for 5-12 hours and then chips are washed. 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.
M-Guide: Build your own arrayer
Array Maker Documentation
Repeatability & Accuracy
Sample Tracking Software
Ngai Lab arrayer , UC Berkeley
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
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
Product Amplification and preparation: What to Print?
MJ Tetrad PCR machine
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 H 2 O 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.
Non - Contact
Syringe-solenoid ink-jet dispenser
Contact (using rigid pin tools, similar to filter array)
Micro spotting pin
Micro Spotting pin
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
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.
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)