We currently have a problem with our qPCR on miRNA. We use the kit nCode miRNA First-Strand cDNA Synthesis for polyadenylation and reverse transcription of miRNA. For qPCR we use our own SYBR Green and our own protocol.
We tested several quantities of miRNA, ranging from 0.5 to 2 µg. The primers used are universal primer (complementary to the poly-A) and a primer complementary to our miRNA. Only during the PCR, our samples do not come out, there are only the baseline. Even adding 10 cycles with our PCR cycles to reach 55 total. We also tested several Tm and several primers miRNA but nothing has changed.
We ask ourselves the question: how to determine the quality of our cDNA?
After extraction of our total RNA, we found their quality with Agilent Bioanalyzer 2100, samples used with a RIN greater than 7.
If anyone has encountered this kind of problem, its help will be welcome.
Edited by preston, 15 March 2011 - 03:15 AM.