Problems with two step RT-PCR - (Apr/26/2012 )
We're currently trying to detect mRNA expressions of GOI in human saliva. We have previously did this in PBMC and have obtained decent results. Hence we tried to do the same in saliva, using the same protocol with the exception of RNA extraction method. Using VILO for cDNA synthesis and Taqman for real time, the mRNA expression of all genes (including 18S as housekeeping gene) with saliva are significantly blunted compared to PBMC. This means that we ended up getting Ct values of >37 for the GOI, which is unreliable. The 18S expression also came out with Ct around 18 for saliva, whereas in PBMC it was around 10. The standard curves for 18S looks ok, however the sensitivity is really poor with the GOI.
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?
18S rRNA expression can be regulated by growth stimuli and vary among cell types depending on proliferation status. So it is possible that 18s rRNA expression is lower in saliva than PBMC.
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/content/64/2/669.full.pdf
Kitty, I had tried really hard without success to extract intact RNA from saliva. What method did you use for the RNA extraction? I am sure you mean using Nanodrop for RNA quantitation not RNA integrity. Could you show a gel picture of your extracted RNA?