Microarray Protocols

Microarray Protocols

Scientists at the OMRF Microarray Research Facility are available to address technical questions about cDNA production, labeling, and hybridizations. Many parameters have been tested and optimized for the particular equipment in our facility. We request that you cite our facility in publications if you find these protocols useful. The following protocols are available for downloading.

Protocols

Succinic Anhydride Slide Blocking Protocol

Direct Cy-Labeling Protocol with Omniscript RT (modified for Ventana Discovery)

Direct Cy-Labeling Protocol with EndoFree RT (modified for Ventana Discovery)

Indirect Cy-Labeling Protocol (OMRF Modified Amersham CyScribe Labeling)

Automated Hybridization Protocol (Ventana Discovery Optimized Protocol)

Validation

Omniscript cDNA Synthesis for Quantitative Real-Time PCR (QRT PCR)

SYBR Green I Quantitative Real Time PCR (QRT PCR) Protocol

Quantitative Real-Time PCR (QRT PCR) Using EndoFree Reverse Transcriptase

Succinic Anhydride Slide Blocking Protocol

OMRF oligonucleotide microarrays are UV-crosslinked but not blocked. They may be blocked individually or as many as 24 blocked at a time.

Materials Needed for a small number of microarrays:

50 ml conical polypropylene tubes with caps (various vendors)

Succinic Anhydride (Aldrich #23969-0 or Sigma #S7626)

1-methyl-2-pyrrolidinone (Aldrich #270458)

Boric Acid (various vendors)

Sodium Hydroxide (various vendors)

0.2 m Filtered MilliQ or Nanopure Water

95% Ethanol (various vendors)

Orbital Shaker

Rack for 50 ml tubes

Hemostat or Tweezers

Centrifuge for 50 ml tubes or compressed nitrogen gas cylinder

0.2 m Filter & Bottle

Diamond tip pen

Materials Needed for a large number of microarrays:

Slide staining rack or tray

Succinic Anhydride (Aldrich #23969-0 or Sigma #S7626)

1-methyl-2-pyrrolidinone (Aldrich #270458)

Boric Acid (various vendors)

Sodium Hydroxide (various vendors)

0.2 micron Filtered MilliQ or Nanopure Water

95% Ethanol (various vendors)

Orbital Shaker

Compressed nitrogen gas cylinder

0.2 m Filter & Bottle

Blocking Procedure for Individual or a Few Slides:

1. Prepare 0.2 M sodium borate (pH 8.0) by dissolving boric acid in water and adjusting the pH to 8.0 with NaOH. Filter sterilize. Store at room temperature.

2. Mark array area using the diamond tip pen by etching the back of the slide to serve later as a guide for the cover slip. Place slides in a container or box for easy access.

3. Working in the fume hood, transfer 31.5 ml of 1-methyl-2-pyrrolidinone to a 50 ml conical tube.

4. Weigh out 0.5 g succinic anhydride and bring it to the fume hood.

5. Add the succinic anhydride to the 1-methyl-2-pyrrolidinone in the 50 ml tube. Cap the tube and gently mix until the flakes dissolve (this takes about 1 minute).

6. Just after the last flak e of succinic anhydride dissolves, add 3.5 ml of 0.2 M sodium borate (pH 8.0) Cap the tube as soon as possible and mix once by inverting.

7. Remove the cap and immediately submerge a slide (or two slides back to back) into the solution. Recap, tap the tube firmly to dislodge any air bubbles attached to the glass, and begin agitation by transferring tubes to a rack on an orbital shaker so that the slides are horizontal and will remain fully submerged. Shake gently for 15 minutes (we use setting 2.5 on our Hoefer "Red-Rotor" shaker that goes from settings 0 to 10).

8. After 15 minutes, use a hemostat or tweezers to transfer each slide to a separate 50 ml tube with ~ 45 mL room temperature water. Cap and agitate on the orbital shaker in the horizontal position for 2 minutes.

9. Transfer each slide to a separate 50 ml tube with ~ 35 ml of 95% ethanol, cap, and gently invert for about 5-10 seconds.

10. Transfer the slides to an empty, clean, dry 50 ml centrifuge tube and cap. Dry the slides by centrifuging them for 5 minutes at ~1000 rpm. Immediately remove slides from tubes at the end of the spin, allow a few seconds for any residual moisture to evaporate, and transfer slide to a clean container for storage.

11. Store slides dry at room temperature until hybridization. The microarrays are now ready for hybridization.

High Throughput Blocking Procedure: We follow the Corning CMT-GAPS II manual, soaking up to 24 slides in a rack and slide staining dish, or in a beaker with the succinic anhydride/ sodium borate solution.

1. Prepare 0.2 M NaBorate (pH 8.0) by dissolving boric acid in water and adjusting the pH to 8.0 with NaOH. Filter sterilize. Store at room temperature.

2. Dissolve 4.0 g succinic anhydride in 252 ml 1-methyl-2-pyrrolidinone in 500 ml beaker.

3. Put slides in slide holding rack.

4. Just after the last flake of succinic anhydride dissolves, add 28 ml 0.2 M sodium borate (pH 8.0).

5. Mix briefly and immediately submerge slides in the solution, tap the tray to dislodge any air bubbles that may be attached to the glass, and begin agitation immediately, continuing for 15 minutes.

6. Pour off the blocking solution, rinse briefly with water, then transfer the rack containing the microarrays to a 2 L beaker of containing room temperature water, tap to remove air bubbles, and soak for 2 minutes.

7. Transfer the rack containing the microarrays and rinse briefly in 95% ethanol.

8. Dry the slides individually using compressed nitrogen. Stor e slides dry at room temperature.

NOTES:

1. Salt crystals from the printing buffer make it possible to visualize the area on the slide printed with DNA before blocking the slides. These crytals are dissolved during blocking. If you are using hand hybridization under glass coverslips, you should etch a line with a diamond-tipped pen on the back of the slide to mark the area containing the DNA before blocking. Thus will make it easy to position a coverslip before hybridization. If you are using an automated hybridization system, the etching is not necessary.

2. Succinic anhydride has a very short half-life. This makes it important to have the slides ready to immerse, the 0.2 M sodium borate ready to pipet, and to work quickly. It is important to begin agitation immediately after adding the slides to the blocking solution to avoid a phenomenon called "comet tails". Comet tails occur when unbound DNA dissolves in the blocking buffer and binds to the glass surface adjacent to where it was spotted, hence forming streaky tails from the spots.

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Cy3 or Cy5-dUTP Direct Labeling Protocol Using Omniscript Reverse Transcriptase

This protocol labels 2 ug of total RNA using Omniscript reverse transcriptase for use in hybridizations on the Ventana Discovery System.

Materials required:

Omniscript RT™ Kit (Qiagen #205113)

Cy3-dUTP (Amersham #PA53032)

Cy5-dUTP (Amersham #PA55022)

2M HEPES (Ambion)

Montage PCR 96-well Cleanup (Millipore #LSKC09604)

10x dNTP mixture (made from 100 mM dNTP commercial stocks

2.5 N NaOH

Cy-3-labeled anchored oligo-dT(20)VN primer (Operon, custom synthesis)

Note: Use RNAse-free tubes and pipet tips

Program a PCR machine for 65oC 5min, 37oC 2 hr, and 37oC 15 minutes.

If a thermocycler is not available, heat one incubator to 65°C and a second incubator to 37°C. Water baths, heat blocks, thermal cyclers, or hybridization ovens (set to stationary) are all suitable for reverse transcription reactions as long as they are capable of maintaining temperature within the suggested range of ±1°C.

cDNA Labeling

1. Mix together in PCR RNase free tube (Ambion#12225):

· 2 mg total RNA

· Nuclease-free water (ex. Gibco #10977) to bring thevolume to 12.5 ml

· 1 ml anchored oligo dT primer from a 500 ng/ml stock concentration. The final [oligo-dT] will be 0.5 mM in 20 ml volume.

Note: With other reverse transcriptases, we have used the same mass of oligo dT(12-18) primer (Invitrogen #18418-012)

2. Heat to 65oC for 5 minutes.

3. Prepare the following reaction mixture and add to the above tube containing the RNA and primer

10X RT buffer 2.0 ml

10x dNTP mix (see below) 2.0 ml

Cy3 or Cy5 dUTP (0.5 nmole) 0.5 ml

40 U RNAse Inhibitor 1.0 ml

4 U Omniscript RT 1.0 ml

10x dNTP mix contains2.5 mM dATP, dCTP, dGTP and 1.5 mM TTP in water.

4. Incubate at 37oC for 2 hours.

5. Add 2 ml 2.5 N NaOH and incubate at 37oC for 15 min.

6. Add 10ml 2M HEPES, vortex, and centrifuge briefly.

Note: At this point the samples may be stored at 4oC for several days.

Clean the labeled cDNA using Montage plates or Microcon spin columns.

Our preliminary experiments indicate that more genes were detected above background on microarrays using the Montage system. The level of expression of genes expressed in both systems was similar.

Montage PCR 96-well Cleanup Procedure

Details and troubleshooting may be found in the Millipore technical bulletin #TN1600EN00 available on-line at http://millipore.com/publications.nsf/docs/TN1600EN00.

1. Add 270 ml water to each well to be used in the plate. You do not have to use an entire plate. Wells not used should be left dry for future use. Once wells are used, do not reuse them for other samples.

2. Add your sample (approximately 32 ml) to the appropriate wells.

3. Place the Montage plate on a vacuum manifold and apply 10 inches mercury vacuum until the wells are visually dry. Turn off the vacuum and allow the plate to reach atmospheric pressure.

4. Add 25 ml water per well. Cover the plate with an adhesive plate seal.

5. Shake the covered plate on a rotating shaking table for 10 minutes at room temperature as fast as possible without allowing the samples to come into contact with the seal (i.e. 1100 rpm on our IKA Shuttler MKS4 shaker).

6. Use pipets to transfer the resuspended nucleic acid to a microfuge tubes. The volume should be between 20-25 ml. If the volume is considerably larger, re-apply the vacuum to the plate to further concentrate those particular sample(s).

Microcon Spin Column Cleanup Procedure.

1. Add 500 ml TE (Ambion#9849) to a Microcon-100 column (Millipore, 42413).

2. Centrifuge for 3 minutes at 5000 x g and discard the flow-through.

3. Add 300 ml TE to your labeled cDNA, and add pipet the solution to the prewashed Microcon-100 column.

4. Centrifuge for 3 minutes, 5000 x g and discard the flow-through.

5. Wash the Microcon column with 400 ml TE. Centrifuge for 3 minutes, 5000 x g and discard the flow-through.

6. Wash the Microcon column again with 400 ml TE. Centrifuge for 3 minutes, 5000 x g and discard the flow-through.

7. Invert column into a fresh microfuge tube and centrifuge for 3 minutes, 5000 x g. Carefully measure the volume of the probe. It should be <20 ml. If it isn’t, put it back in the Microcon and centrifuge for another minute at 5000 x g before rechecking the volume.

To check the percent incorporation of cy3 and cy5 use the formula from the following web site http://www.pangloss.com/seidel/Protocols/perc ent_inc.html

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Cy3 or Cy5-dUTP Direct Labeling Protocol Using EndoFree Reverse Transcriptase

This protocol labels 2 ug of total RNA using EndoFree reverse transcriptase for use in hybridizations on the Ventana Discovery System.

Materials required:

EndoFree RT™ Kit (Ambion #1740)

Cy3-dUTP (Amersham #NEL578)

Cy5-dUTP (Amersham #NEL579)

500 mM EDTA (Ambion#9260G)

1 M Tris-HCl (pH 8) (Ambion#9855G)

Montage PCR 96-well Cleanup (Millipore #LSKC09604)

Note: Use RNAse-free tubes and pipet tips

Program a PCR machine for 70oC 5min, 42oC 65 m in, and 65oC 60 minutes.

If a thermocycler is not available, heat one incubator to 50°C and a second incubator to 70°C Water baths, heat blocks, thermal cyclers, or hybridization ovens (set to stationary) are all suitable for reverse transcription reactions as long as they are capable of maintaining temperature within the suggested range ±1°C.

cDNA Labeling

1. Mix together in PCR RNase free tube (Ambion#12225):

· 2 mg total RNA

· Add nuclease-free water (ex. Gibco #10977) to bring the volume to 7 ml

· 1 ml of anchored oligo dT primer supplied in the kit. The final [oligo-dT] will be 0.5 mM in 20 ml volume.

Note: With other reverse transcriptases, we have used 1 ul of a 500 ng/ml oligo dT12-18 primer) (Invitrogen Cat #18418-012)

2. Heat to 70oC for 10 minutes. Transfer quickly to 50oC and incubate for 10 min.

3. Prepare the following reaction mix and incubate at 50°C for 10 min

10X RT buffer 2.0 ml

dCTP Solution 2.0 ml

dGTP Solution 2.0 ml

dATP Solution 2.0 ml

dTTP Solution 0.6 ml

Cy3 or Cy5 dUTP Solution 0.5 ml

RNAse Inhibitor 2.0 ml

EndoFreeRT 1.0 ml

4. Pipette 12.1 µl of the reaction mixture (from step 3) into the RNA / primer mixture (from step 2). Pipette solutions quickly to avoid cooling below ~48°C.

5. Incubate at 50oC for 2 hours.
6. Add 5 ml 500mM EDTA (Ambion#9260G), vortex, and centrifuge quickly.

7. Add 10 ml 1M NaOH, vortex, and centrifuge briefly. (EDTA and NaOH may be pre-mixed immediatedly before addition).

8. Incubate at 65oC for 1 hour.

9. Cool to room temperature and add 25 ml of 1M Tris-HCl (pH 8) (Ambion#9855G), vortex, and briefly centrifuge the samples.

Note: At this point the samples may be stored at 4oC for several days.

Clean the labeled cDNA using Montage plates or Microcon spin columns.

Our preliminary experiments indicate that more genes were expressed above background on microarrays using the Montage system. The level of expression of genes expressed in both systems was similar.

Montage PCR 96-well Cleanup Procedure

Details and troubleshooting may be found in the Millipore technical bulletin #TN1600EN00 available on-line at http://millipore.com/publications.nsf/docs/TN1600EN00.

1. Add 240 ml water to each well to be used in the plate. You do not have to use an entire plate. Wells not used should be left dry for future use. Once wells are used, do not reuse them for other samples.

2. Add your sample (approximately 60 ml) to the appropriate wells.

3. Place the Montage plate on a vacuum manifold and apply 10 inches mercury vacuum until the wells are visually dry. Turn off the vacuum and allow the plate to reach atmospheric pressure.

4. Add 25 ml water per well. Cover the plate with an adhesive plate seal.