RT PCR - (Jan/18/2006 )
When I prepare samples for reverse - transcription, and estimate the RNA, the ratios indicate that the quality of my RNA is good. I then synthesize the cDNA, make the PCR cocktail and run the samples. I get erratic results. One sample will work, another won't. Any suggestions on where the problem may lie?
just on the off chance your spec is flaky, have you tried a gel to spot-check a few samples?
my other best guess would be a problem with the cDNA synthesis
first question: how long between the time you prep the RNA and the time you make the cDNA?
second question: would you please give us a few details about your cDNA synthesis protocol?