Does Taq have reverse transcriptase activity? - (Aug/31/2005 )
I didn't know that there were so many actin pseudogenes but i suspected it. That's why actin is a good gene to check genomic contamination in a RT minus control. I mean it's easier to amplifly b actin from genomic DNA when it is present as contaminant in low amounts. In other case it could pass unnoticed.
But some of these pseudogenes may be transcribed. Check some intronic PCRs
And how are you sure that your DNA was completely digested? I'd say the evidence in your RT neg control shows that it wasn't.
LeserattePD
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How long was the incubation time for DNase, sometimes you have to increase the incubation time.
[quote=sirin,Sep 7 2005, 06:07 AM]
And how are you sure that your DNA was completely digested? I'd say the evidence in your RT neg control shows that it wasn't.
LeserattePD
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How long was the incubation time for DNase, sometimes you have to increase the incubation time.
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I digested half an hour with 4U of DNase I at 37.
At first time I thought that it was an incomplete digestion of genomic. Then I digested purified genomic DNA with higher amounts of DNase and I saw that 4U were enough to degradate 2 ug of gDNA (which is much more than the amount of contaminant genomic in RNA sample)
PCR is very sensitive. So how would you be able to prove that all your DNA is really degraded? Normally you would do his e.g. by PCR! So I don't think that any referee will accept your 'proof' for the existence of reverse-transcriptase activity.
I agree that I haven't shown that my RNA sample is genomic-Free. However I'm not sure that PCR of the minus RT control against a no coding region, is the ultimate proof for absence of genomic. Actually, in genomic DNA we find a single copy of a gen within a huge amount of nucleic acid. So if I wouldn't see amplification with intronic primers I couldn't be so sure about lack of genomic.
you say "I'm not sure", but you haven't produced any data to demonstrate whether it is true or not
In my experience proof or lack of amplification of an intronic region of a non-housekeeping gene is the best way to show whether or not there is gDNA contamination
If you got amplification of an intronic region of a RNA sample there's no doubt that we are in presence of genomic contamination. But what if you've got no amplification. Would you say RNA is genomic-Free? I wouldn't say that. I'd just say that my primers weren't able to amplificate DNA contamination in my specifical PCR conditions. If that negative result would be together with a positve control (e.g. amplification of a equivalent amount of purified genomic DNA in the same PCR conditions) I could say that there was genomic.
Hi, This is an interesting topic... I am not sure that you can ever say that there is NO gDNA contamination.
The point is that the amount of gDNA in each sample is below the level of detection with your primers/PCR conditions, or that you are using primers that span an intron and therefore do not amplify genomic DNA. Any time you can, you should use intron-spanning primers for RT-PCR. This is hard to do with housekeeping genes, because as discussed above there are alot of pseudogenes for these (GAPDH beta-actin etc.)
Because pseudogenes are present for the housekeeping genes, in order to compare between samples, you must confirm that gDNA does not affect the results with your control gene (beta-actin). If you can see amplimers in the no RT control then you have to re-DNAse your samples because you have enough background gDNA present to affect the results...
Re-DNAse, then maybe use less RNA for RT reaction---this will reduce the amount of contaminating gDNA as well.
You should PCR the RT- and RT+ samples at the same time with the same number of cycles etc, then as long as no bands are detected in the RT- you can trust the RT+ result. This does not mean that there is NO gDNA, only that it is not present in sufficient quantitiy to affect the result, and that is what is important.
Anyway that is my 2 cents... HTH
I think you might be interested in the following article:
Martel F, Grundemann D, Schomig E. A simple method for elimination of false positive results in RT-PCR.
J Biochem Mol Biol. 2002 Mar 31;35(2):248-50.