qPCR from HEK cells transfected with Plasmid - (Mar/01/2013 )
Hi,
I want to do qPCR on a gene which is encoded on a plasmid. I have HEK cells transfected and subsequently isolated RNA and digested the extract with DNase. Before reverse transcription I perform a control pcr to check there is no more plasmid present. This is how it should be... Is this possible? How big is the risk that there is still some plasmid left and therefore invalidate my qPCR results? Any tricks or special things I have to take care of?
Thanks
You want to do a reverse-transcription qPCR on a gene, that is encoded on plasmid?
You isolate RNA, treat with DNase, transcribe and include RT- control (ideally for every sample).
If your RT- is negative, then the signal you got is only from mRNA/cDNA.
For initial testing of DNase efficiency you do this with only few samples, any samples, not with the experimental setup.
Thank you, I know how qPCR works. Anyway, my concerns were more about that plasmid DNA is not efficiently removed during RNA isolation and DNase can have problems with degrading plasmid DNA, at least this is what I read elsewhere. I wanted to know if there are any specific tricks to avoid these troubles.
Cheers
In your question, you state that there is no plasmid present. As Trof indicated, you should demonstrate this in a more quantitative way: Perform qPCR on your samples and in parallel the same samples where you left out the RT enzyme. I've personally done some experiments like these, including on-column DNAse digestion, and my Ct-values were highly correlated between +RT and -RT samples, indicating plasmid contamination. I'm going to try this kit from Agilent next: http://www.chem.agil...5989-1097EN.pdf
Has anyone tried it?
cellthetruth on Sat Mar 2 17:01:07 2013 said:
Thank you, I know how qPCR works. Anyway, my concerns were more about that plasmid DNA is not efficiently removed during RNA isolation and DNase can have problems with degrading plasmid DNA, at least this is what I read elsewhere. I wanted to know if there are any specific tricks to avoid these troubles.
You probably know how qPCR works, but seems you don't know how RT- works, otherwise you wouldn't ask about that again.
I was simply trying to understand clearly what are you planning to do, by the way.
We were doing experiments on cells transfected by plasmid, isolated RNA on Qiagen columns with on-column DNAse treatment (which removes most, but not all DNA) and then additionaly treated by Turbo DNA-Free kit from Ambion as we treat all our RNAs with that.
RT- control was always negative, so we didn't have any problems with DNase not digesting plasmid using our setup.