Target Discovery and Validation Reviews and Protocols

68 Beaty et al.
3.1.3. Labeling of mRNA
1. For each test or reference sample, in a 1.5-mL Eppendorf tube on ice add the
following:
• 50 µg of total RNA (or 2 µg of mRNA).
• 5 µg of RT primer, and RNase-free H2O to a total volume of 15 µL.
2. Heat samples at 70°C for 10 min and then cool on ice 3 min, quick spin at 4°C and
hold on ice.
3. On ice, add 3 µL of Cy5 dUTP or Cy3 dUTP to test and reference samples, respectively
(see Note 5). Tap gently to mix then quick spin.
4. On ice, add 12.1 µL of RT Mastermix (6 µL of 5X First-Strand buffer, 3 µL of 0.1 M
DTT, 0.6 µL of 50X dNTPs, 0.5 µL of RNasin, and 2 µL of Superscript II). Tap
gently to mix and then quick spin and incubate 42°C for 1 h.
5. Thereafter, add an additional 1 µL of Superscript II, mix gently, quick-spin, and
incubate at 42°C an additional 1 h.
6. Cool on ice and add 3 µL of 0.5 M EDTA stop solution, mix gently, and quick spin.
3.1.4. Hybridization of Labeled Sample
1. Combine Cy5-labeled test and Cy3-labeled reference samples, together with 450 µL
of TE7.4 , into a Microcon-30 filter, and spin 12,000g for 9–12 min at room temperature
in a microcentrifuge; retained volume should be approx 20 µL (see Note 6);
discard the flow-through.
2. Add an additional 500 µL of TE7.4 , spin at 12,000g for 9–12 min, and discard the
flow-through (see Note 7).
3. Add an additional 450 µL of TE7.4 ,2 µL of polyA RNA stock solution, 2 µL
of yeast tRNA stock solution, and 20 µL of human Cot-1 stock solution (see
Note 8).
4. Spin 12,000g for 9–12 min and discard the flow-through. The retained volume
should be less than 32 µL.
5. Invert Microcon-30 filter into a new Eppendorf tube, and spin at 12,000g for 1 min
to recover labeled cDNA sample.
6. Transfer the labeled cDNA mixture to a screw-top microcentrifuge tube, determine
the sample volume by using a pipet, and increase the volume to 32 µL by
adding ddH2O. 7. Add 6.8 µL of 20X SSC and mix. Add 1.2 µL of 10X SDS, mix gently to avoid
forming bubbles.
8. Boil the hybridization mixture for 2 min, incubate at 37°C for 20 min, and then
quick spin.
9. Carefully pipet hybridization mixture onto DNA microarray and overlay with a
22- × 60-mm glass cover slip (see Note 9).
10. Overlay cover slip with several small droplets of 3X SSC (totaling 20 µL) to provide
hydration and then enclose and seal slide in hybridization chamber. Incubate
at 65°C for 16–18 h (see Note 10).