DNA Protocols

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DNA Micromicroarray 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.

Notes:

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.

Notes:

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).

Materials needed:

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

27.0 µl

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.

Notes:

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 other side.
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.

Materials needed:

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

H2O 76.0

Taq DNA polymerase (5 U/µl) 1.0

100 µl

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 (TeleChem)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.

Notes:

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 (TeleChem) are free of contaminants that clog spotting pins.

Materials needed:

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 (TeleChem, 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).

Notes:

Use SuperAldehyde Substrates (TeleChem).
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.

Materials needed:

SuperAldehyde Micromicroarray Substrates (TeleChem).
Robotic printing device (Cartesian or TeleChem).

VI. Hybridization.

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.

Notes:

Hybridization cassettes (TeleChem).
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.

Materials needed:

Hyridization cassettes (TeleChem).
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.

Notes:

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).

Materials needed:

Fluorescent laser scanning device.

VI. References.

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.
mark@arrayit.com

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