Services - Microarray Protocols
DNA Microarray Protocols
(Courtesy of Mark Schena and Ron Davis, Stanford University, 1997)
I. Preparation of total RNA from cultured human cells.
- Obtain a confluent flask containing 50 ml of cultured human cells (e.g. Jurkat line J25).
- Set up 5 flasks (162 cm2) containing 100 ml per flask of RPMI media.1
- Split confluent cells 1:10 and grow 48 hrs.
- Add inducer (e.g. phorbol ester) and grow an additional 24 hrs.
- Transfer cells (500 ml total) to a 500 ml centrifuge bottle.
- Pellet cells by centrifugation in a JA-10 rotor for 5 min at 3,000 rpm.
- Remove and discard media.
- Resuspend cell pellet in 10 ml phosphate buffered saline (PBS).
- Transfer to a 15 ml conical tube.
- Pellet cells by centrifugation in a clinical centrifuge for 5 min at 1,000 rpm.
- Remove and discard PBS.
- Freeze cell pellet (~5 x 108 cells) in liquid nitrogen and store at -80°C.
- Obtain cell pellet (~5 x 108 cells) from -80°C.
- Add 12 ml of GTC solution2 and resuspend pellet by vortexing.
- Draw the cell lysate through a 19-gauge needle 10 times using a 12 cc syringe.3
- Set up six ultracentrifuge tubes4 containing 3.0 ml each cesium chloride solution.5
- Overlay 2 ml of the GTC cell lysate onto the CsCl layer of each tube.
- Pellet the RNA by centrifugation in an SW50.1 rotor at 35,000 rpm for 12 hrs at 25°C.
- Pour off and discard the supernatant by inverting each tube.6
- Dry tubes in an inverted position for 60 min on a layer of paper towels.
- Add 100 µl of 1X TE7 to the bottom of each tube.8
- Heat for 1 min at 65°C to aid in dissolving the pelleted RNA.
- Pipet vigorously to dislodge and dissolve pelleted RNA.
- Combine the six 100 µl RNA samples.
- Use 0.4 ml fresh 1X TE7 to isolate residual RNA from each tube.
- Extract the 1.0 ml RNA sample twice with 0.5 ml phenol9 to remove residual protein.
- Extract twice with ether to remove trace organics.
- Divide the RNA sample into two 500 µl aliquots in eppendorf tubes.
- Add 50 µl 2.5 M sodium acetate (depc-treated) and 1.0 ml ethanol to each tube.
- Pellet the RNA by centrifugation for 15 min at 25°C in a microfuge.
- Remove and discard the supernatant.
- Dry the RNA pellet in a speedvac.
- Resuspend the RNA pellet in 0.4 ml 1X TE.7
- Concentration should be 7 mg/ml for a total yield of 3 mg RNA per 500 ml cells.
- RPMI media is supplemented with 10% fetal bovine serum, 100 µg/ml streptomycin and 500 U/ml penicillin.
- Dissolve 60 g guanidine thiocyanate, 0.5 g sodium N-lauroylsarcosine, 5.0 ml 1M sodium citrate in 100 ml of H2O. Sterile filter. Add 0.5 ml ß-mercaptoethanol.
- Shearing chromosomal DNA increases RNA yield.
- Use Beckman Ultra-Clear tubes 13 x 51 mm (#344057).
- Dissolve 95 g CsCl and 20 ml 0.5 M EDTA (pH=8.0) in 100 ml of H2O. Sterilize by filtration. Add diethyl pyrocarbonate (depc) to 0.1%. Autoclave.
- DO NOT allow the supernatant to drain back onto the RNA pellet.
- 1X TE is treated with 0.1% depc to inactivate ribonucleases.
- Avoid the inner sides of the tube which may contain trace ribonuclease.
- Phenol solution contains phenol/chloroform/isoamyl alcohol (25:24:1 v/v).
II. Preparation of polyA+ mRNA from total human RNA.
- Thaw total RNA (~7 mg/ml) stored at -80°C.
- Mix 150 µl RNA (~1 mg), 150 µl 2X binding buffer, and 55 µl Qiagen Oligotex-dT resin.
- Heat 3 min at 65°C to denature the RNA.
- Cool to room temperature 10 min to allow annealing of mRNA to resin.
- Pellet the resin containing bound mRNA by spinning for 2 min in a microfuge.
- Resuspend the resin in 600 µl of wash buffer by vigorous vortexing.
- Pellet the resin by spinning for 2 min in a microfuge.1
- Resuspend the resin in 600 µl of wash buffer.
- Transfer to a Qiagen spin column.
- Spin out the wash buffer by centrifugation for 30 sec in a microfuge.
- Resuspend the resin in 33 µl of 80°C elution buffer.2
- Spin for 30 sec and transfer the eluant containing the mRNA to a new tube.
- Elute the mRNA with two additional 33 µl volumes of elution buffer as above.
- Combine the three 33 µl mRNA fractions.
- Add 10 µl of 2.5 M sodium acetate3 and 220 µl of 100% ethanol.
- Spin out the mRNA by centrifugation for 15 min at 25°C in a microfuge.
- Dry the mRNA pellet in a speedvac.
- Resuspend the pellet in 11 µl of 1X TE (pH=8.0).3
- Use 1.0 µl for quantitation.4
- Dilute to 1.0 µg/µl.
- Yield should be 20-25 µg mRNA from 1.0 mg of total RNA.
- Carry out the first wash in an eppendorf tube (ie. by “batch wash”) to remove particles and debris that clog the spin column.
- Heat the eppendorf tube containing the spin column to 80°C for 30 sec to assist in mRNA elution.
- Solutions treated with 0.1% diethyl pyrocarbonate to inactivate ribonucleases.
- Mix 1.0 µl in 0.5 ml TE and determine the absorbance at 260 nm (OD1.0=40µg/ml).
- Qiagen Oligotex mRNA Midi Kit (#70042).
III. Probe preparation by single-round reverse transcription.
Reagent Volume (µl)
Total mRNA (1.0 µg/µl)1 5.0
Control mRNA cocktail (0.5 ng/µl)2 1.0
Oligo-dT 21mer (1.0 µg/µl) 4.0
H2O (DEPC-treated) 17.0
Denature mRNA 3 min at 65°C. Anneal Oligo-dT to mRNA 10 min at 25°C.
5X First Strand Buffer 10.0
10X DTT (0.1 M) 5.0
RNase Block (20 U/µl) 1.5
dATP, dGTP, dTTP Cocktail (25 mM each) 1.0
dCTP (1 mM) 2.0
Cy3-dCTP (1 mM)3 2.0
SuperScript II Reverse Transcriptase (200 U/µl) 1.5
50.0 µl total
- Reverse transcribe 2 hrs at 37°C.
- Add 5.0 µl of 2.5 M sodium acetate and 110 µl 100% ethanol at 25°C.4
- Centrifuge for 15 min at 25°C in a microfuge to pellet cDNA/mRNA hybrids.5
- Remove and discard supernatant and carefully wash pellet with 0.5 ml 80% ethanol.6
- Dry pellet in a speedvac and resuspend in 10.0 µl 1X TE (pH 8.0).7,8
- Boil sample 3 min to denature cDNA/mRNA hybrids. Chill on ice immediately.
- Add 2.5 µl 1N NaOH and incubate 10 min at 37°C to degrade the mRNA.
- Neutralize the cDNA mixture by adding 2.5 µl 1 M Tris-Cl pH 6.8 and 2.0 µl 1M HCl.
- Add 1.7 µl 2.5 M sodium acetate and 37 µl 100% ethanol.
- Centrifuge for 15 min at full speed in a microfuge to pellet the cDNA.5
- Remove and discard supernatant and wash pellet with 0.5 ml 80% ethanol.6
- Dry pellet in a speedvac and resuspend in 6.5 µl H2O.
- Add 2.5 µl 20X SSC and 1.0 µl 2% SDS.
- Heat at 65°C for 0.5 min to dissolve probe mixture.
- Centrifuge for 2 min in a microfuge at high speed to pellet trace debris.9
- Transfer supernatant to a new tube.
- Probe concentration ~0.5 µg/µl per fluor in 5X SSC and 0.2% SDS.
- Total mRNA purified from total RNA using Oligotex-dT (see part II).
- Cocktail contains a dilution series of Arabidopsis mRNAs transcribed in vitro.
- To label mRNA with other fluors, substitute Fl12- or Cy5-dCTP in the reaction.
- Chilling or use of >2 volumes of ethanol results in precipitation of free label.
- Pellet product on one side of the tube, then remove supernatant from the otherside.
- To prevent loss of pellet, centrifuge 1 min before removing 80% ethanol.
- Product often smears up the side of the tube. Resuspend thoroughly!
- Resuspend the fluorescein-, Cy3- or Cy5-labeled products in 10 µl total volume.
- Tiny particles interfere with hybridization, which is carried out under a cover slip.
- StrataScript RT-PCR kit (Stratagene #200420).
- Oligo-dT 21 mer (treated with 0.1% depc to inactivate ribonucleases).
- 100 mM dATP, dCTP, dGTP, dTTP (Pharmacia #27-2050, -2060, -2070, -2080).
- 1 mM Cy3-dCTP (Amersham #PA53021).
- 1 mM Cy5-dCTP (Amersham #PA55021).
- 1 mM fluorescein-12-dCTP (DuPont #NEL-424). Do Not use FluorX-dCTP (Amersham), which reduces signal by more than 10-fold.
- SuperScript II RNase H- Reverse Transcriptase (Gibco BRL #18064-014).
IV. PCR amplification and product purification.
Reagent Volume (µl)
10X PCR buffer (15 mM Mg2+) 10.0
dNTP cocktail (2 mM each) 10.0
Primer 1 (100 pmole/µl)1,2 1.0
Primer 2 (100 pmole/µl)1,2 1.0
Miniprep plasmid DNA (10 ng/µl)3 1.0
Taq DNA polymerase (5 U/µl) 1.0
- Amplify targets in a 96-well format using 30 rounds of PCR (94°C, 30 sec; 55°C, 30 sec; 72°C, 60 sec).
- Purify using the MicroarrayIt 96-well PCR purification kit (Arrayit)4.
- Elute with 100 µl of 0.1X TE pH = 8.0.
- Dry to completion in a speedvac.
- Resuspend each PCR product in 7.5 µl 5X SSC (0.3-1.0 mg/ml DNA).
- Transfer to a flat bottom 384-well plate (Nunc) for microarraying.
- Use of generic primer pairs (~21mers) to vector sequences allows high-throughput.
- Use primers with a 5’ amino-modification to increase linking to glass surface.
- Alkaline lysis method. The 96-well REAL prep (Qiagen) facilitates high-throughput.
- MicroarrayIt PCR products (Arrayit) are free of contaminants that clog spotting pins.
- 96-well REAL prep alkaline lysis kit (Qiagen #SQ811 and #19504).
- PCR primers modified with a 5’-amino-modifier C6 (Glen Research #10-1906-90).
- GeneAmp PCR system 9600 (Perkin Elmer #N801-0001).
- MicroAmp 96-well PCR reaction plates (Perkin Elmer #N801-0560).
- Taq DNA polymerase (Stratagene #600139).
- MicroarrayIt 96-well PCR purification kit (Arrayit, http://arrayit.com)
- Flat-bottom 384-well plates (Nunc #242765).
V. Micromicroarraying and Slide Processing.
- Obtain SuperAldehyde substrates.1
- Print cDNAs or oligos using a robotic printing device.2
- Allow printed micromicroarrays to dry overnight in a slide box.3
- Soak slides twice in 0.2%SDS for 2 min at room temperature with vigorous agitation.5
- Soak slides twice in dH2O for 2 min at room temperature with vigorous agitation.4 Transfer slides into dH2O at 95-100°C for 2 min to allow DNA denaturation.
- Allow slides to dry thoroughly at room temperature (~5 min).
- Transfer slides into a sodium borohydride solution for 5 min at room temperature to reduce free aldehydes.5
- Rinse slides three times in 0.2% SDS for 1 min each at room temperature.
- Rinse slides once in dH2O for 1 min at room temperature
- Submerge slides in dH2O at 95-100°C for 2 seconds.6
- Allow the slides to air dry and store in the dark at 25°C (stable for >6 months).
- Use SuperAldehyde Substrates (Arrayit).
- Microarray elements will dessicate during microarraying and the crystaline DNA spots can be observed under a dissecting microscope to identify missing features.
- Drying increases crosslinking efficiency. Several days or more is OK.
- Removes salt and unbound DNA.
- Dissolve 1.0 g NaBH4 in 300 ml phosphate buffered saline (PBS). Add 100 ml 100% ethanol to reduce bubbling. Prepare JUST PRIOR to use!
- Heating the slides greatly aids in the drying process.
- SuperAldehyde Micromicroarray Substrates (Arrayit).
- Robotic printing device (Cartesian or Arrayit).
- Place the micromicroarray in a hybridization cassette.1
- Add 3.0 µl of 0.1% SDS to the bottom of the microarray for humidification.2
- Aliquot 5.0 µl of the fluorescent probe (see Part III) onto the edge of the micromicroarray.
- Cover the probe droplet with a 22 mm square glass cover slip using forceps.3,4
- Seal the cassette containing the microarray with a glass plate and metal clamps.1
- Submerge the hybridization cassette in a water bath set at 62°C.
- Hybridize for 6 hrs at 62°C.
- Following hybridization, remove the microarray from the hybridization cassette.
- Place the microarray immediately into a beaker containing 400 ml 1XSSC and 0.1% SDS.5
- Wash the microarray by gentle buffer agitation for 5 min at room temperature.6
- Transfer the microarray to a second beaker containing 400 ml 0.1XSSC and 0.1% SDS.
- Wash the microarray by gentle buffer agitation for 5 min at room temperature.6
- Rinse the microarray briefly in a third beaker containing 0.1X SSC to remove the SDS.
- Allow the microarrays to air dry.7
- Scan for fluorescence emission.
- Hybridization cassettes (Arrayit).
- Aliquot the 0.1% SDS onto the frosted labeling surface of the microscope slide.
- Cover slips must be dust- and particle-free to allow even seating on the microarray.
- Air bubbles trapped under the cover slip exit after several minutes at 62°C.
- Use a 600 ml pyrex beaker containing a magnetic stir bar.
- Buffer agitation accomplished by placing the beaker on a stir plate.
- Cy3 or Cy5 microarrays are scanned dry. Scan for fluorescein in 0.1X SSC.
- Hyridization cassettes (Arrayit).
- 22 mm square cover slips (Corning).
V. Scanning and image analysis.
- Place slide onto the stage of a confocal fluorescent scanner or CCD-based detection device.1
- Scan the microarray for fluorescein emission.2, 3
- For two-color experiments, scan the same microarray for Cy3 or Cy5 emission.4, 5
- Save the image as a TIFF file.
- Import the file into NIH image to convert to a pseudocolor scale.
- Analyze TIFF file data with AIS/BMS software.
- Commercial systems from PerkinElmer, Axon, and others.
- Scan at a PMT setting of ~5.5 for current confocal device.Lower PMT settings enable linear detection of the abundant transcripts.
- Slight adjustments of the PMT allow exact intensity-matching of the two-color scans.
- Fluorescein (494/525 nm), Cy3 (552/570 nm), Cy5 (643/667 nm).
- Fluorescent laser scanning device.
- Schena, M. and R.W. Davis (1997). Parallel Analysis with Biological Chips. in PCR Methods Manual, Academic Press (San Diego), in press.
- Heller, R.A., Schena, M., Chai, A., Shalon, D., Bedilion, T., Gilmore, J., Woolley, D.E., and R.W. Davis (1997). Discovery and analysis of inflammatory disease-related genes using cDNA micromicroarrays. Proc. Natl. Acad. Sci. USA 94, 2150-2155.
- Schena, M., Shalon, D., Heller, R., Chai, A., Brown, P.O., and R.W. Davis (1996). Parallel human genome analysis: Micromicroarray-based expression monitoring of 1,000 genes. Proc. Natl. Acad. Sci. USA 93, 10614-10619.
- Schena, M. (1996). Genome analysis with gene expression micromicroarrays. BioEssays 18, 427-431.
- Shalon, D., Smith, S.J., and P.O. Brown (1996). Genome Research 6, 639-645.
- Schena, M., Shalon, D., Davis, R.W. and P.O. Brown (1995). Quantitative monitoring of gene expression patterns with a complementary DNA micromicroarray. Science 270, 467-470.
VII. Technical questions.
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Recommended Equipment and Reagents
SpotBot® 3 Personal Microarrayers
InnoScan® Microarray Scanners
SpotLight 2 Microarray Scanners
Microarray Hybridization Cassettes
High Throughput Wash Station
Microarray High-Speed Centrifuge
Protein Printing Buffer
BlockIt Blocking Buffer
Microarray Air Jet
Microarray Cleanroom Wipes
PCR Purification Kits
Micro-Total RNA Extraction Kit
MiniAmp mRNA Amplification Kit
Indirect Amino Allyl Fluorescent Labeling Kit
Universal Reference mRNA
Green540 and Red640 Reactive Fluorescent Dyes
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