We are trying to reverse transcribe large RNAs that have substantial secondary and tertiary structure. We don't get much product with standard reverse transcriptases. The thermostable reverse transcriptases don't appear to perform any better, possibly because the higher temperatures promote chemical or RNase-mediated RNA hydrolysis. So heat does not appear to be an option to reduce secondary structure. Have any ResearchGate members had success reverse transcribing large RNAs with significant secondary/tertiary structure? Can you supplement buffers with nucleic acid denaturing agents and retain high fidelity reverse transcription? Is there a method for preparing reverse transcriptase buffers free of contaminating nucleases? Any help/insight you can provide to answers these questions would be greatly appreciated.
Amadeo Parissenti
Professor, Laurentian University and the Northern Ontario School of Medicine
Dear professor Parissenti I would like to hep you, but I should ask you about some details, please.
1. Your final goal with RNA? (cloning, expression, sequencing, any oth?)
2. Is it eucaryotic RNA?
3. Which size (approximately) does it have?
4. Is it single gene RNA or different RNAs pull?
5. Does it have isoforms i.e. alternative NM/XM in NCBI?
I work with RNA and probably may be able to help you.
In any case I am very interested in this topic.
Thanks for responding, Andrei: We are trying to reverse transcribe the entire length of a series of related human transcripts with gene specific primers. Some transcripts are greater than 4 kbp with significant predicted secondary structures. We believe that it is the secondary structure that is interfering with full length reverse transcription. Hope this clarifies our problem. We would be very grateful, if you might have some thoughts on how to reduce nucleic acid secondary structure, while not affecting the activity of the reverse transcriptases. We have no issue with degradation of the total RNA input when using thermostable reverse transcriptases at temperatures lower than 65 degrees C, but we don't get full length reverse transcripts with the very large RNAs.
Yes, your problem is real. I planed to propose you high temperature-based transcriptases. However I have colleague who can help. He can try to help, of course. I ask him soon. He is biologist and work in development of molecular biological reagents. Including thermostable polymerases and transcriptases with. He clone each ferment by himself using domestic design for mRNA sequence. May be he was touched with problem like you.
Dear Dr. Parissenti, a mean that you can try to solve your problem as next:
1. First, if available, enrich mRNA fraction by any way, without RNA degradation (Bioanalyzer or Experion control is needed);
2. Use at least +42oC compatible reverse transcriptase. The temperature balance between RNA degradation and secondary structure solving is needed.
3. Double the amount of RNA to reaction. For example 2 ug insteed 1 ug according to manufacturer manual.
4. Add betaine and DMSO. About 1M final concentration of betaine and 3-8% DMSO.
Unfortubately in our experience we have not significant effect in such cases, but we have it.
I hope my advice will help you.
Dear Andrei: Thank you kindly for your thoughtful reply. We were thinking on supplementing the RT buffers for the thermostable reverse transcriptase with betaine, DMSO, or DMF. If you are recommending this approach as well, then we'll definitely try it. We were going to try a range of concentrations for these agents, so we'll make sure to span the concentrations you are recommending. We'll also try a variety of reaction temperatures to push the limits of the thermostable reverse transcriptase.
Thanks again for your kind suggestions and we'll let you know if we are successful! Wish us luck!
Amadeo
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