Methods Digest, Vol 69, Issue 1
(by dnreisman from ufl.edu)
Fri Feb 4 15:08:38 EST 2011
Viral rna likely forms hairpin structures. We have a similar problem when we tried to sequence GC rich regions. Try using superscript 3. This Reverse transcriptase can work at higher temperatures (not 42C like most other RTs)..... we typical used 50-58C for our RT reactions. A this temps the secondary RNA tends to go away allowing the RT reaction to work.
Hope this helps
University of Florida
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1. problems with one step RT-PCR (Dunowska, Magda)
Date: Fri, 4 Feb 2011 16:55:25 +1300
From: "Dunowska, Magda" <M.Dunowska from massey.ac.nz>
Subject: problems with one step RT-PCR
To: "methods from magpie.bio.indiana.edu" <methods from magpie.bio.indiana.edu>
Cc: "Stickney, Alison" <A.Stickney from massey.ac.nz>
<92FDFD8B26EB6542B1E1BF017BB998D16990116E0B from TUR-EXCHMBX.massey.ac.nz>
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Hi, my student has run into some problems with the reverese transcriptase PCR (RT PCR) assay that I can't quite understand...we are trying to amplify viral RNA with published primers designed to amplify 166 bp product. The published protocol used FRET probes. We tried this and got no signal. We then decided to start from scratch and check the primers with cDNA made from viral RNA (in a separate step) with a SYTO dye assay using the AccuMelt (Quanta) PCR mix. We got a nice amplification with single melting peaks for all positive samples and the right size single band when the reactions were checked on a gel. So, we had an amplifiable template and primers that recognised the template. We then added the reverse transcriptase to the reaction and included an additional RT step before amplification (50 deg C for 10 minutes). This seems to have killed the assay - we got no amplification at all. What I couldn't understand is that not only we got no signal from tubes with RNA, but we!
also got no amplification in one tube with cDNA (that worked just fine in the same reaction without the RT enzyme). We also tried three different commercial kits designed for a one-step RT-PCR and got no amplification with any of them. So, we can amplify cDNA in a PCR reaction, but when we try to amplify RNA or the same cDNA in the RT-PCR reaction we get no amplification at all. I would have thought that even if the RT reaction didn't work we should get amplification of the cDNA in the RT-PCR assay...Has anybody come across such a bizarre thing? It seems like the RT enzyme (we've tried two different enzymes from two different manufacturers) interferes with amplification. I used the same enzymes/buffers to amplify other viral RNA targets with no problems...Any ideas of what might be going on and how to fix it (short of running the assays in a two-step format which would be much more expensive and time-consuming)?
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