I have been doing this method for about 4 months now and have had mixed
success. Sometimes I get ladders, other times not and after many control
experiemnts to nail down the offending step(s) I have little to report. I
DO know that high quality of the input RNA is absolutely essential to the
success of the assay. Try Operon's primers, they're 1/30th the cost of
GenHunter's and "work" just as well. I feel the first two or three cycles
are the most critical in the PCR step and have increased the denaturing
time to 2 minutes prior to the first annealing and extension.