How concerned do I have to be about a qRT-PCR experiment in that RNA was not treated with DNAse prior RT and primer pairs are within one exon?
Is this a no-no-no or is this usually fine since cDNA transcripts should usually be many more then the two genes on the DNA?
Wondering about past and future designs...
Thanks for your advise :D
It's something that's worth worrying about if you aren't running appropriate controls. You can include a no-RT control to determine how much of the signal you observe is coming from the genomic DNA itself. This can establish the 'floor' of your assay. It's also good to include a no-RNA control to establish the floor for primer self-amplification. Ideally, you'd also want to determine the linear range and efficiency of the reaction to see whether your observations are in the linear range.
I would say this is a no-no-no, but this really depends on your extraction protocol and the expression level of your transcript. Some kits claim to have almost pure extraction of RNA, whereas some may yield very high levels of DNA contamination.
If trying to detect a low-abundance RNA molecule, DNA contamination will have a huge impact on your analysis and the majority of your results may actually be reflective of DNA contamination and not transcription rates.
As Brian mentioned, you can process several samples without RT and check for the level of expression compared to your actual results. If you're detection is multiple CTs lower in all of your samples with RT as compared to your RT minus controls, you are probably okay. If the CTs are closer than that, I would probably suggest you throw out the data.
In future experiments it is never a bad idea to span a junction. Always run a control without RT to check for DNA contamination.
Hi, I agree with Michael Glen Becker and Brian Bower advices.
Hi, I agree with Michael Glen Becker and Brian Bower advices. But i would like to say something else.
In my practice with total RNA extraction from cells and tissues (fresh frozen samples in liquid nitrogen or even native samples immediately after surgery) DNA contamination with silica-based approaches (columns or sorbents) approximately 4-15%! on the base on fluorimetry data.
In the case of gene expression analysis where you target gene shows at least Cq=25 and less it is not a big problem to have 4-5% of genomic DNA. Of course in the case of Cq=30-35 you have to control DNA contamination more precisely.
I do not reccomend you use DNAse treatment for gene expression analysis, but it could be quiete usefull in the case of clonong and some oth. procedures. Tha fact is that DNAse treatment can increase the rate of your RNA degradation. At keast you have to spend more time....and time it is crucial parameter when you work with RNA.
My advices:
- use Qubit or Quantus for dsDNA contamination control.
- do not use DNAse treatment.
- control time.
Unfortunately even exon span qPCR experiments it is possible to amplyfy genomic DNA because of abberant pseudogenes of "retropseudogenes".
Hope it helps. Good luck.
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