Problems with two step RT-PCR
Posted 25 April 2012 - 11:54 PM
We've also eliminated the possibility of inhibitor within the Taqman real time step by spiking the saliva cDNA with other cDNA source and found that the Ct obtained is correlated with the amount of other cDNA we put it (not saliva cDNA). This makes us think that the cDNA synthesis step is the fault. We have also measured the integrity of RNA by nanodrop and denaturing agarose gel and they both look fine.
I guess my questions are:
1.) Across different human cells, should the 18S expression be similar?
2.) Is there a way to check whether the RNA has successfully turned into cDNA in the first step? (we don't have Ribogreen or anything like that in the lab)
3.) Does anyone have an idea what could cause the RNA not being turned into cDNA, however still ok in the real time PCR?
Posted 26 April 2012 - 07:35 PM
There is no good way to test whether RNA has been successfully converted to cDNA besides PCR amplification.
There are studies showing that reverse transcriptase can inhibit PCR reaction when template concentration is low. See this paper http://aem.asm.org/c.../2/669.full.pdf
Posted 06 June 2012 - 05:45 PM