Protocols

The quality of microarrays is heavily dependent on the quality of the RNA used to make the labeled target. Therefore, RNA isolation methods and RNA quality control assays are critical. We recommend the protocol below so that prepared RNA samples produce valid and reliable microarray and PCR results. The basic procedure is to (1) isolate total RNA using TRI REAGENT (MRC, Inc. Cincinnati, OH). One can visit the MRC website for the most recent protocols (select RNA Isolation). Note that RNAzol and RNAzol B reagents are based on an older patent and their use may result in higher DNA contamination levels. (2) Treat the isolated RNA with a DNAse/Proteinase K step, which digests any contaminating DNA and protein. Contaminating DNA causes variable microarray and PCR results, and contaminating protein can inhibit labeling and cause RNA degradation. (3) Extract the RNA with TRI REAGENT LS to remove the digested DNA and proteins. We also recommend isolating total RNA rather than poly(A)+ RNA for use in microarrays. The protocols described here can also be viewed at http://microarray.uc.edu.

Protocol 1: Isolation of total RNA from tissue and cells

Method

  1. For tissues, using pre-chilled long tweezers, place tissue directly into 1 ml of liquid N2 in a 15-ml, round-bottom tube. Keep the tube at -80°C (without the cap for 24 hr). Frozen tissue can remain at -80°C indefinitely.
  2. Transfer the tissue into a fresh 15-ml, round-bottom tube containing TRI REAGENT. We recommend 1 ml TRI REAGENT per 50 mg tissue. Increasing the amount of tissue increases the chance of DNA and protein contamination.
  3. Immediately homogenize the tissue with Kontes homogenizer and pestle. Be sure that all of the tissue is homogenized. Rinse 3X with water between samples.
  4. Transfer 750 µl of the homogenate to a 1.5-ml microfuge tube. If thawing homogenates, briefly place in room temperature water bath and swirl.
  5. Spin homogenates at 12,000 x g for 10 min. Transfer the supernatants into new microfuge tubes.
    • For cultured cells, remove the culture medium by aspiration, add at least 1 ml TRI REAGENT per 10 cm2 cells. Pass the cell lysate several times through pipette to complete cell lysis.
  6. For tissue and cell lysates, incubate for 5 min at RT.
  7. It is beneficial to perform phase separation with bromochloropropane (BCP, MRC # BP 151) instead of chloroform. Use 100 µl of BCP per ml of the reagent. The use of BCP instead of chloroform improves the quality of the isolated RNA. In addition, BCP is less toxic and less volatile than chloroform. If chloroform is used, the volume of chloroform should always be 20% of the initial volume of the reagent. Vortex for a full 15 sec and incubate for 3 min at room temperature. Using a higher volume of chloroform will increase DNA contamination.
  8. Centrifuge 12,000 x g for 15 min at 4°C.
  9. Transfer aqueous phase to a new centrifuge tube. Do not disturb the interphase.
  10. Add one-half volume isopropanol and incubate samples 10 min at room temperature (not longer, proteins will precipitate). Always add one-half volume isopropanol to the initial volume of the reagent or salts and proteins may co-precipitate.
  11. Pellet RNA 12,000 x g for 10 min at 4°C.
  12. Remove supernatant. Caution: Pellet may be loose. Quickly spin and remove residue.
  13. Add 1 ml 75% ethanol to pellet, vortex, and centrifuge 7,500 x g for 5 min at 4°C.
  14. Remove Ethanol. Caution: Pellet may be loose. Quickly spin and remove residue with 10-µl pipette.
  15. Air dry on ice for 5 min (do not dry in hood).
  16. Re-dissolve completely in 87 µl of RNase-free/low endonuclease water.
  17. DNase/Proteinase K Treatment: Add 10 µl of DNase buffer, and 3 µl (6 U) of TURBO DNase (Ambion #2238). Mix and incubate for 15 min at 37°C. Add 4 µl of Proteinase K (New England BioLabs #P8102S; 20 mg/ml). Mix and incubate for 30 min at 37°C.
  18. Use TRI REAGENT LS to extract RNA. Add RNase-free/low endonuclease water to makeup the volume to 0.25 ml.
  19. Add 0.75 ml of TRI REAGENT LS and shake vigorously for 15 sec. Store the lysates for 5 min at room temperature.
  20. Add 0.1 ml of BCP, cover the sample tightly and shake vigorously. Store the mixture at room temperature for 8 min. Centrifuge the mixture at 12,000 x g for 15 min at 4°C.
  21. Transfer the top aqueous phase into a fresh tube. Precipitate the RNA by adding 0.5 ml isopropyl alcohol. Store the samples at room temperature for 8 min and centrifuge at 12,000 x g for 8 min at 4°C.
  22. Remove the supernatant and wash 1 ml of 75% ethanol. Centrifuge at 12,000 x g for 8 min at 4°C. Carefully discard the supernatant and air dry the pellet for 5 min.
  23. Dissolve the pellet in a small volume of RNase-free/low endonuclease water.
  24. For labeling for microarray analysis, the final concentration of RNA should be 2 µg/µl.
  25. To determine accurate concentrations and 260/280 absorbance ratios, the RNA should be assayed on a NanoDrop ND-1000 (Wilmington, DE); and to determine RNA quality (degree of degradation), the RNA should be assayed on an Agilent 2100 Bioanalyzer (Foster City, CA).
  26. For both the NanoDrop and Agilent 2100 Bioanalyzer analyses, remove 1 µg of the RNA sample and dilute to 0.1 µg/µl in TE. The 260/280 absorbance ratio should be above 1.8.

Notes: Precautions when working with RNA

Although for optimal labeling we recommend 20-30 µg of total RNA, typically, when total RNA amounts are 10 µg or greater (5 µg or greater when of superior quality), the RNA can be efficiently converted to cDNA and labeled by an indirect method using the protocol described below. The protocol is a slightly modified version of the Brown Lab (Stanford University) protocol available on their website (http://cmgm.stanford.edu/pbrown/protocols/amino-allyl.htm). The Brown lab protocol is based on a protocol developed by DeRisi (1). The original protocol is available at http://www.microarray.org.

Protocol 2: cDNA synthesis and indirect amino-allyl labeling

Reagents and Solutions

Method: Day 1

Reverse Transcription (RT) Reaction: For optimal labeling, 20-30 µg of total RNA (2-3 µg/µl) is required per reaction. As little as 5 µg of total RNA can be used if of high quality.

1. To anneal the oligo(dT) to the RNA template, add the following for each reaction:

oligo (dT) 5 µg of 2 µg/µl 2.5 µl
nuclease free-water   5.5 µl
total RNA 20 µg of 2 µg/µl 10.0 µl
  TOTAL 18.0 µl

When there are multiple reactions, prepare a master mix of oligo(dT) and RNase-free/low endonuclease water.

2. To denature, heat to 70°C for 10 min. Cool on ice for 5 min.

3. Add 11.6 µl of RT Master Mix (below) to each Cy3 and Cy5 reaction. For multiple reactions, calculate the master mix volume to accommodate an extra 1-2 reactions.

RT Master Mix (based on Superscript III RT kit, Invitrogen #18080-044):

First Strand Buffer (in Superscript III RT kit) 5X 6.0 µl
dNTP Stock Solution (25mM of each deoxynucleotide) 50X 0.6 µl
DTT (in Superscript III RT kit) 0.1 M 3.0 µl
Superscript III RT (Invitrogen; Cat#18080-044) 200 U/µl 1.5 µl
RNAsin (Fisher; Cat#FP2221) 40 U/µl 0.5 µl
  TOTAL 11.6 µl

4. Incubate reaction at 50°C for 1 hr.

RNA hydrolysis:

5. Degrade RNA by addition of 15 µl of 0.1N NaOH. Incubate at 70°C for 10min.

6. Cool to room temperature. Neutralize by addition of 15 µl 0.1N HCl.

Ethanol Precipitation:

7. Transfer reaction mixtures to a 1.5-ml tube.

8. Add 6 µl 3M sodium acetate, pH 5.2.

9. Add 150 µl cold absolute ethanol.

10. Store at -20°C overnight.

Day 2

1. Centrifuge tubes at 12,000 x g for 15 min at 4°C.

2. Pour off supernatant. Add 750 µl cold 80% ethanol; flick the tube.

3. Centrifuge tubes at 12,000 x g for 10 min at 4°C.

4. Repeat steps 2 and 3.

5. Vacuum dry samples for 10 min.

Dye coupling:

6. Add 5µl of coupling buffer and 5µl water.

7. Mix well by vortexing. Incubate at 42°C for 10 min.

8. Mix well by vortexing. Centrifuge to collect the sample.

9. Mix the sample by pipetting and dissolve Cy3/Cy5 dye with this solution. Mix well by pipetting.

10. Incubate 1 hr at RT in the dark. Mix by vortexing every 15-30 min.

Cleanup with Qia-Quick PCR purification kit (Qiagen #28104 or #28106):

11. Add 250 µl Buffer PB, add 35 µl of 0.1 M sodium acetate, pH 5.2, to each reaction tube. Vortex and centrifuge.

12. Apply the mixture to Qia-quick column and centrifuge at 12,000 x g for 1 min. Decant flow-through.

13. Add 750 µl Buffer PE and centrifuge at 12,000 x g for 1 min. Decant flow-through. Centrifuge at 12,000 x g for 1 min.

14. Transfer spin unit to new 1.5-ml microfuge tube.

15. Add 35 µl Buffer EB to center of filter and incubate for 5 min at room temperature. Centrifuge at 12,000 x g for 1 min.

16. Vacuum-dry the samples. Samples are ready for hybridization.

Notes

Total RNA samples that are degraded and/or in amounts of less than 5 µg require one to two rounds of amplification in order to attain the required amount of labeled target. We follow the protocol described in the accompanying literature with the Amino Allyl MessageAmpTM kit (catalog #1753) from Ambion (Austin, TX) for target amplification. In our hands, the kit produces approximately 50-120 µg of amplified RNA (aRNA) starting with 2 µg of total RNA (10 µg of each labeled aRNA is used per slide). As little as 100 ng of total RNA can be amplified with two rounds. The kit is based on a modified Eberwine procedure (2), in which double stranded DNA is generated from the RNA template by reverse transcriptase and an oligo(dT) primer fused to a T7 promoter followed by DNA polymerase. Amino allyl UTP is incorporated into aRNA during the amplification process by T7 polymerase. The cyanine dye coupling is carried out as described above for cDNA dye coupling, except that the 5-µl volume of water is not added to the reaction. The aRNA is added to the coupling buffer at a 2 µg/µl concentration and then directly used to suspend the cyanine dye. It has been shown that the aRNA is an accurate representation of the original total cellular RNA (3).

Protocol 3: Microarray slide production

Method

Printing oligonucleotides:

1. The 70-mer oligonucleotides are suspended in 3XSSC (30 µM) and printed on aminosilane-coated slides (Cell Associates CSA-100 C).

UV-crosslinking and slide storage:

2. The printed slides are laid array-side-up in a Stratalinker (Stratagene, La Jolla, CA) and irradiated at 6000 (x100) µjoules, i.e., 60 mJ.

3. The slides are stored at room temperature under vacuum.

Notes

The mouse 70-mer oligonucleotide library (Version 3.0) from Operon Biotechnologies, Inc. (Huntsville, AL) representing a total of 24,878 annotated genes and 32,829 transcripts was used in the studies described here. The 70-mer oligonucleotides are spotted at 22°C and 65-85% relative humidity at ~100 µm center-spacing using a high-speed robotic machine (OmniGrid-100 model; GeneMachines; San Carlos, CA) using Stealth pins (SMP3 pins) from Telechem (Sunnyvale, CA) mounted in a 48-pin head. Spot volumes are 0.5 nl and spot diameters are 75-85 µm.

Protocol 4: Microarray hybridization and wash conditions (4):

Method

Prehybridization:

1. Prehybridization buffer: 5x SSC, 0.1% SDS and 1% BSA (Sigma #B-4287). Heat to 50°C while stirring.

2. Slides to be analyzed are placed in a staining jar; prehybridization buffer is added, and incubation is carried out at 48°C for 45-60 min.

3. The slides are washed by dipping up and down approximately 10 times in two different staining jars of deionized water. Excess water is removed by shaking the slide rack up and down two times.

4. The slides are then dipped in an up and down motion approximately 10 times at room-temperature in isopropanol and spun dried. The slides are used immediately after prehybridization (less than 1 hr) as hybridization efficiency decreases rapidly if the slides are allowed to dry for more than that time.

Hybridization:

5. 2X hybridization buffer: 50% formamide, 10X SSC and 0.2% SDS. Incubate the solution until it reaches 48°C.

6. The Cy3 and Cy5 labeled targets are suspended in 9 µl water, and the mixture is heated to 95°C for 3 min to denature, and centrifuged at maximum angular velocity for 1 min.

7. The following are added to each tube in order to block non-specific hybridization. Make a master-mix with the following ingredients for each tube:

8. 21 µl 2X hybridization buffer that has been pre-heated to 48°C is added to the target mixture, mixed well, and centrifuged.

9. The labeled target is applied to a pre-hybridized microarray slide and covered with a 22 x 60 mm glass cover slip.

10. The slide is placed in a sealed hybridization chamber (Corning, Acton, MA), and 12 µl water is added to the small reservoirs at each end of the chamber.

11. The sealed chamber is placed in a 48°C water bath and incubated for 40-60 hr (5).

Post-hybridization washes:

12. The array is removed from the hybridization chamber with care taken not to disturb the coverslip.

13. The slide is placed in a rack for a staining dish containing 1X SSC, 0.1% SDS, and 0.1 mM DTT at 48°C.

14. The coverslip is gently removed while the slide is in solution and agitated for 15 min.

15. The slides are transferred to a staining dish containing 0.1X SSC, 0.1% SDS, and 0.1 mM DTT at 48°C and agitated for 5 min.

16. Repeat step 15 two more times.

17. The slides are transferred to a staining dish containing 0.1X SSC and 0.1 mM DTT at room-temperature and agitated for 5 min.

18. Repeat step 17.

19. Slides are spun dried.

Notes

TROUBLESHOOTING
We have found that when a microarray slide is judged a failure as determined visually or by statistical analysis, in approximately 90% of the cases the failure is ultimately due to lower quality RNA. Thus, it cannot be overstressed how important it is to carefully isolate the RNA and carry out quality control assays on the purified RNA. Other far less common sources of failure include missing or merged spots on the microarray slide, insufficient prehybridization or washing, leaks, and other mistakes due to human error.

REFERENCES

  1. DeRisi, J., Penland, L., Brown, P. O., Bittner, M. L., Meltzer, P. S., Ray, M., Chen, Y., Su, Y. A., and Trent, J. M. (1996) Nat Genet 14, 457-460
  2. Van Gelder, R. N., von Zastrow, M. E., Yool, A., Dement, W. C., Barchas, J. D., and Eberwine, J. H. (1990) Proc Natl Acad Sci U S A 87, 1663-1667
  3. Iscove, N. N., Barbara, M., Gu, M., Gibson, M., Modi, C., and Winegarden, N. (2002) Nat Biotechnol 20, 940-943
  4. Hegde, P., Qi, R., Abernathy, K., Gay, C., Dharap, S., Gaspard, R., Hughes, J. E., Snesrud, E., Lee, N., and Quackenbush, J. (2000) Biotechniques 29, 548-550, 552-544, 556 passim
  5. Sartor, M., Schwanekamp, J., Halbleib, D., Mohamed, I., Karyala, S., Medvedovic, M., and Tomlinson, C. R. (2004) Biotechniques 36, 790-796