RT-PCR inconsistant results - (Jul/22/2008 )
Since starting my phd 6 months ago, i have been screening cell lines using RT-PCR. I have sorted out contamination issues(eventually), however, sometimes my sample come up positive and sometimes it is negative.
I have used different batches of cells to isolate mRNA, and made several batches of cDNA from the same mRNA stock too.
Of 3 separate reactions i did today, the first was negative, the second was positive and the third was negative again.
For each reaction i do, i run a no-template-control and a postive control. I also run the negative cDNA (no reverse transcriptase) for the sample too. Negative controls are clear, and the positive control is always strong.
hope you can help me - this is driving me insane!
Have you checked if there is residual RNase contamination in your purified RNA (incubating an aliquot of your RNA in caCl2 containing buffer for a few minutes), which could give you inconsistent results. If you have RNase contamination, you may want to try the AquaRNA RNase decontamination protocol.