I ran the negative control RT reaction (no RT enzyme) using random hexamers and oligo dT primers. I then used that same sample for qPCR of three different genes in fish skeletal muscle. What is perplexing to me is that I got amplification of two of the genes but not the third in my negative controls (the amplification is around 10% of the amplification in samples which did have reverse transcription).
I checked two different muscle RNA samples and they both gave me different amounts of amplification of these two genes while the same third gene showed no amplification. When I run a gel of these samples I get the expected size band for my primers. This is how I know its not genomic DNA I'm getting: I designed all my primers to span an intron, and I'm not getting a larger than expected band in my gels.
The only thing I can think of to explain this is that the taq polymerase in the TaqMan master mix is somehow polymerizing straight from RNA. and its doing this in a template specific manner i.e. two of the genes amplify but the third doesnt in the negative controls. Is that possible?
Dear Ammon ...
In my opinion there can be two possibilites that an explain such behaviour
1) some RNA or cDNA comtamination may have come in some of your reagents and as you mentioned that the amounts in negative controls are low...check this possibility carefully
2) second things is what you are thinking...RT activity of Taq polymerase....there are a good numbers of papers on RT activity of Taq as well as many other DNA polymerases (Taq polymerase does this in the presence of Mn inons...check these in you assay).....and they are not very recent....check few of them... and set appropriate contorls to justify your points....take a look at some papers on the lonks given below
http://www.springerlink.com/content/uw7g92ypgnju3buu/
http://www2.mrc-lmb.cam.ac.uk/archive/papers/CP90-07.pdf
http://www.roboklon.de/pdf/LTR_1.pdf
and also see the attached file
bye
Dear Ammon,
Hi did i understand correctly that you have 3 singel genes seperately or you use a multiplex PCR???.
How about negative control for primers (no cDNA or RNA) in de mix???.
It is possible that primer dimers can be formed with specific primes and remaining unused primers from RT mix.
Tthan you would see primer dimers band around 2 or three times of size of your specific primers.
By sequencing of the DNA product you saw in agarose gel (which you judged as specific by size) you can see differences between cary- over contamination or primer dimer formation.
Success
Thank you all for your responses. My primers do span exon boundaries. I've run the reaction on two different RNA samples and got some variability in the amount of amplification in my negative controls. I guess it is possible that I got some contamination from other amplifications of two of my genes but not the third and to differing degrees in the two different RNA samples I used, but that seems very unlikely. What seems to me to be the most plausible explanation I've heard is that the TaqMan polymerase is having some reverse transcription activity and the mRNA for the three different genes are surviving the high temperatures during the various stages of the reactions to differing degrees. I talked to appliedbiosystems and they think its very unlikely but I dont know. I'll look into the possibility of contamination from other pcr reactions.
Another question: does the RNA get completely degraded during the initial 10 minute 95 degree incubation?
In the past one of my collegues found that our pippettors were contaminated, resulting in false positive signals from the negative control.
Check the contamination of your primer pairs 1 and 2 (using all the components but no template).
Is your genes have been already describe in your fish specie? If there is no previous work that validate the annotation of your genome, it is possible that you have a problem of assembly in your DNA database. That could explain an amplification due to genomic contamination that could give the "good size product".
Am unclear too. So you do have a primer/probe system? not just two primers?
As others have suggested, some control to check reagent contamination would be appropriate--set up a reaction with no RNA/cDNA at all--i.e. "water template". If you get the correct-sized product from this, you likely have product contamination in some reagent--or possibly a pipettor. Do this at least in triplicate--if the contamination level is low, it might not show up in every reaction. Another thought is to use genomic DNA as template at the same time as you do the "water template' test. If there is a product from genomic DNA and not the water template, then there may be a pseudogene in genomic DNA that amplifies with your "mRNA-specific" primers.
It is quite possible that any residual RNA will be at least badly degraded by 95 for 10 minute heating at the start of the qPCR. Heating RNA in the presence of Mg++, such as present in your qPCR reaction, does cause rapid degradation--so that would seem to make the possibility of RT activity by Taq an even more unlikely explanation.
Great thanks everybody. I've checked my reagents and it does look like there is some PCR product contamination from two of my genes. Since the relative signal varies from plate to plate I think maybe the contamination is being introduced by my pipetors or through the tubes I use to make my reaction mixtures rather than the stock solutions themselves.
Good job! Yes, it could be your pipettors, (clean them often, use filter tips, be meticulous about using one set of pipettors that are only used for "pre-PCR" activities--that never go near PCR products.) It could also be just very low levels of contamination of some reagent--so that the number of molecules in any particular aliquot may vary around a low number--say 0 to 4--which will appear to give variable signals in your "no template' reactions.
Run a negative control (no template); not no RT emzyme. If you are using SYBR green check the melt curve. At what cylce number dopes your extraneous product appear? Is it beyond cycle 28-30?
Hi Ammon,
I had the similar problem. My -RT reactions started to show bands of cDNA size. After a lot of PCR I found out that there was some cDNA contamination in the PCR mixture and a in the Primer stock solution. I ended up ordering new primers and now everything is fine.
With primer design it is usually best if you can have it spanning 2 exons. Thus only transcribed info will show up in PCR (not genomic). now if that is the case and you are still getting a +ve, it could be that you have a reverse transcriptase contamination in one of your reagents. thus amplifying your RNA (false positive).
Hi,
I had the same problem with my -RT samples which showed expected size bands using primers that are either 2 or 3 exon spanning. This indicates that it is not a genomic DNA contamination.
I also run non template controls which did not show any ct values nor a band when I ran a gel. In this case I dont think I have a contamination problem but rather DNA ploymerase amplifying the RNA or pseudogenes that are amplified. Any other thoughts? What can I do to pevent this?
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