I have been working on an RNA virus. In order to synthesize cDNA from the total RNA isolated from the infected plant material, I used a specific reverse primer near the end portion of the viral RNA (more specifically oligo dT primer), M-MuLV reverse transcriptase enzyme, and carried out the reaction at 42C for an hour. I confirmed the cDNA by amplifying it with a set of gene specific primers, and it produced the amplicon of the expected size. But when I was trying to amplify it with different set of primers which was supposed to give a larger amplicon, the reaction failed multiple times. All I could amplify was upto 1.3 kb only. But I am expecting cDNA of 2 kb + size. Good Suggestions regarding this will come handy for my experiment. Thank you in advance
It may be that you have an issue with viral RNA secondary structure stalling synthesis (https://www.thermofisher.com/uk/en/home/life-science/cloning/cloning-learning-center/invitrogen-school-of-molecular-biology/rt-education/reverse-transcription-setup.html). You might consider undertaking synthesis at a higher temperature, perhaps with an engineered reverse transcriptase to reduce this issue (see https://www.neb.com/applications/cloning-and-synthetic-biology/dna-preparation/reverse-transcription-cdna-synthesis).
Craig S Wilding actually we have a standardized temperature of 65C in our lab for the breakage of the secondary structures. So we proceeded accordingly. So do you think instead of 65C, we should increase the temperature by one or two degrees? or shall we increase the incubation time from 5 minutes to 10 minutes or so? And, with your second reference, I have tried with Superscript RT from TaKaRa as well. According to their manual, it can efficiently synthesize upto 10kb cDNA. I used their exact protocol. However, the same results I obtained.
I was thinking that your reaction temperature (42oC) is quite low so whilst you may have broken secondary structure at 65oC, the RNA may subsequently refold at such a low temperature. Some MMLV reverse transcriptases can be used up to 55oC and this might help.
The other thing to consider is the lability of RNA and if your RNA is not either fresh, or a freshly defrosted aliquot, you may not be getting full-length cDNAs
The RNA I used was fresh one. Exactly I can tell you, it was just one day old when I performed cDNA synthesis from the RNA. And the temperature related information was not known to me. So in that case, if I go for 65C for 10 minutes followed by 45C or 50C for the first strand synthesis, the reaction may workout. But should I proceed with the same RNA or should I use newly extracted RNA? The RNA I am having is exactly 12 days old stored at -80C. Craig S Wilding
If it was fresh frozen and never freeze thawed then it should be as good as fresh RNA
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