For mRNA capture, 20 μl lysate was transferred to Turbocapture (Qiagen) plates coated with oligo dT. Following a 60-minute incubation at room temperature, unbound lysate was removed by inverting the plates onto a highly absorbent towel followed by centrifugation at 1000 rpm for one minute. First-strand cDNA was prepared from the mRNA by adding 5 μl master mix consisting of # units M-MLV reverse transcriptase and # umol/l of each dNTP. Plates were incubated at 37°C for 90 minutes. Probes were annealed to the first-strand cDNA using 5ul Probe Anneal master mix, which contains 100 femtomole of each probe in 1× Taq ligase buffer. Denaturation was accomplished by incubating the plates at 95°C for 2 minutes and then decreasing the temperature from 70°C to 40°C over a 6-hour period. Plates were then inverted onto an absorbent towel and spun at 1,000 RPM for 1 minute to remove unbound probe.
