Has anyone had any problems with their negative controls for some microRNA Taqman assays? With a few assays, replacing RNA with water for the RT reaction still results in qPCR amplification and a generated CT value. This is definitely not due to the water or reverse transcriptase being contaminated with RNA – I have checked this. I am wondering whether if for some assays, the stem-looped primers can be amplified and act as targets for the probe in the qPCR? Does anyone heat denature and snap-cool the miR RT primers prior to use?
Interesting, we never had this problem before. How did you check for contamination? Aside, from using fresh water or a new enzyme vial, what else did you do? Did you check the rt primers. My personal opinion is that you most likely have a contamination, but haven't found the source yet. Although, theoretically, the stem loop primers may under certain condition generate false product I find this unlikely; the Taqman probe has only limited, partial, complementarity to the rt primer and second, even if it can bind along its entire length, I don't expect a perfect complementarity; in that case the PCR reaction will likely not work. Just a thought.
- Badges
- Science topic