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 David Reisman University of Florida -----Original Message----- From: [email protected] [mailto:[email protected]] On Behalf Of [email protected] Sent: Friday, February 04, 2011 12:09 PM To: [email protected] Subject: Methods Digest, Vol 69, Issue 1 Send Methods mailing list submissions to [email protected] To subscribe or unsubscribe via the World Wide Web, visit http://www.bio.net/biomail/listinfo/methods or, via email, send a message with subject or body 'help' to [email protected] You can reach the person managing the list at [email protected] When replying, please edit your Subject line so it is more specific than "Re: Contents of Methods digest..." Today's Topics: 1. problems with one step RT-PCR (Dunowska, Magda) ---------------------------------------------------------------------- Message: 1 Date: Fri, 4 Feb 2011 16:55:25 +1300 From: "Dunowska, Magda" <[email protected]> Subject: problems with one step RT-PCR To: "[email protected]" <[email protected]> Cc: "Stickney, Alison" <[email protected]> Message-ID: <92fdfd8b26eb6542b1e1bf017bb998d16990116...@tur-exchmbx.massey.ac.nz> Content-Type: text/plain; charset="us-ascii" 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)? Thanks, Magda ------------------------------ _______________________________________________ Methods mailing list [email protected] http://www.bio.net/biomail/listinfo/methods End of Methods Digest, Vol 69, Issue 1 ************************************** _______________________________________________ Methods mailing list [email protected] http://www.bio.net/biomail/listinfo/methods
