dpo

Hi Trof, thanks for the suggestion. Unfortunately, the old lot is finished (also at the company) so we can't do side by side comparison. The fact that we aliqout the RNA in single use tubes and that we see an increase in copy number for this oncogene in the two different RNA dilutions, makes it hard for me to believe that it's due to selective degradation. If it would only have happened in one of the two RNA dilutions, degradation would also been my guess, although I would then expect that the copy number would decrease instead of increase. Theoretically, partial degradation may remove some secondary structures that are inhibiting cDNA synthesis, so one could speculate on a mechanism like this resulting in a lower Ct and higher copy numbers.

I think the only option will be to order a new lot of this kit and hope this is working more similar to the old lot than the one we have now. I'm working in a diagnostics lab and it's not so easy to change protocols, it requires extensive validation. One normally hopes/sees that different lot numbers behave similarly, but apparently that's not always the case.

You can check out the plates from Nunc with UpCell Surface (http://www.nuncbrand...e.aspx?ID=11850). Although I have never used these plates, it may come in handy for your purpose. Apparently, shifting the plates to temperatures lower than 32 °C reduces adhesion to the plates and your cells detach without any other intervention. Of course you have to judge whether this temperature shift is permitted in the assay you are planning.