Iīm doing RT-PCR for viral RNA detection for subsequent sequencing (of the cDNA product). But I need to do a nested PCR reaction using the RT-PCR amplified product. If donīt, there is no detectable product. FOr the first reaction Iīm using ONE-STEP RT-PCR form QUIAGEN, but for the nested PCR Iīm using Invitrogen reactives.
Well, everything was working quite well until recently when suddenly negative controls looked positive (amplification product) but only in nested PCR which is highly sensitive. First I thought it was the water I was using, or perhaps stock reaction tubes were allready contaminated from beginning. To adress these hypothesis i set up the following assays:
1. New reaction tubes using new water as a negative control, and as samples the water (RNAse free) tubes I was using for the last PCR assays that went wrong.
2. Stock reaction tubes using as samples molecular reactive grade 1 water RNAse free, and new. (Stock reaction tubes are tubes I leave ready for use, i mean you just have to add 5 microliters of RNA for first PCR reaction, or 3 microliters from RT-PCR product for nested)
3. Excellent (or at least thatīs what I believe) laboratory practices.
The results were the following (Iīll write only NESTED PCR results as there are not detectable or visible cDNA in the first RT-PCR):
1. nested negative control: there was an almost invisible band, but still there it was. (this is the only tube that does not comes from the RT-PCR product)
2. negative control that comes from the first PCR reaction: amplification band.
3. Water tubes that were being tested also were positive: amplification band.
4. Stock tube also: amplification band
From 2 to 3 there was a high intensity amplificaiton band.
Conclusion: water tubes were contaminated so they were discarded.
But result number one was positive and it was preprared with a totally new and pure, etc... water tube (for reaction mix and as sample). From this, I thought maybe primers were contaminated, and also because the signal was almost not visible. Usually contamination products are not as intense as real amplification products.
What do you think?? how can I know if the contamination is in the primers dilution, without using a new set of liofilized primers for testing (thatīs not an option in this moment) ?
I thought, making a new dilution, but what if contamination comes from the highly concentrated stock of rehydrated primers?
What about using other primers for the same nested template but other region? .... no.... they will probably have different annealing temperatures, so it might not work.
Thank you very much.