RNA reverse transcription riddle - (Jun/24/2009 )
My plan is to RT RNA to get cDNA and PCR the cDNA to get the DNA I am interested in. At the first time of RT attempt using Promega Improm II RT, I got a cDNA yield of ~650ng/ul. Before I tried using the cDNA as template, I used a plasmid DNA vector to optimize PCR conditions with my designed primers. The plasmid contains a gene fragment that only has 60bp short in the middle of the sequence comparing to the gene I am interested in. Although the PCR also went well on my cDNA (with 8% DMSO, will talk about this later), the yield was bit low. Then the cDNA was finished and I made new cDNA following exactly the same method and it was when my nightmare began.
The subsequent PCR I did with my newly made cDNAs never produced the entire size as the very first one I had. I then used my forward primer with another reverse primer that both of them target on the first 900bp (not GC rich at all, so without 8% DMSO) of my 2300bp gene fragment and another forward primer with my reverse that both target the last 1750bp (72% GC rich, did with 8% DMSO). The former gave a nice PCR product with plasmid DNA nothing with cDNA and the latter always gave a clear band with right size.
I checked my RNA sequence (~5000bp long) again and found out that it might be too long for the RT to work on till the end. Therefore I changed my RT to Promega AMV (no PCR product for any combination of primers at all) and MMLV, the latter of which is supposed to RT fragments >10kb, but it also showed the same outcome as those done with Improm II.
Know I am really puzzled and don’t know what to do. I checked the troubleshooting suggestions and got advices as lowering
Can I get some advices from you guys? Thank you all so so much!!!
I do not have experience reverse transcribing such a long RNA so I will give you my advice based on what I get from your explanation. It seems to me that you have a PCR assay that works (you can amplify your plasmid DNA without any problems), so your problem is the RT reaction. One way to get around this is to use gene specific primers (instead of random primers) in your RT reaction. By placing the gene-specific primer at position within your gene such that you reverse transcribe no more than 1 to 2 kb at a time (you could even use multiple gene specific primers in your RT reaction) you may be able to get around this problem.
Firstly thank you very much for your quick reply and help. I also think the problem is with my RT reaction. The primer I've been using for my RT is oligodT (it is supposed to bind to the polyA tail of RNA and primes whatever strand it binds to). From the test experiments I have done by far, I can see that the RT had problem just at ~600bp before/downstream of the start codon of my gene. I have also thought about using gene-specific primer for my RT and wonder if I can just simply use the reverse primer of my PCR as my RT primer too, because theoretically it is 100% complementary right? This would shorten RT's job for ~1700bp.
Does anyone have a comment on this idea?
A mayby very stupid question but after the cDNA synthesis did you get rid of the RNA (hydrolysis or RNase H and A) and purified the cDNA using precipitation or a silica column? Because your cDNA yield of 650ng/?µl is very high.
No I did not purify the cDNA after RT. I only set running temperature at the end (70C for 15min) to inactivate the enzymes. I am not sure if the RNA was degraded. However, I ran both the RNA and cDNA on 1% agarose gel and what I saw was 2 faint bands plus a much intenser fat band at the bottom, which I think is the degraded RNA. The cDNA lane also showed the 2 faint bands with the same size as the ones I saw in RNA lane plus an intense band right lower than the 18s band. So clearly there was RNA residues in my cDNA solution.
In order to get rid of the RNA, is simply adding RNase good enough? Many thanks for your idea and help.
Mayby the FAT band are oligo-dt primers? becasue RNA does not degrade (much) after 15minutes at 75°C.
Normally it's not needed to degrade (or remove) the RNA still present after the RT reaction but if you want to be sure that the RT reaction worked I would advise to digest the RNA using RNase A.
The only problem with this is that in order to be sure that you have generated cDNA you should have generated enough to visualize it on gel.
Starting with 1µg of totalRNA (see Promega Improm II RT) will only yield 10-50ng of cDNA when using oligo-dt primers. This should be enough but you can always start with more totalRNA offcourse.
When this all works out I would use the specific primers to PCR the sequence of choice. Problem with this is that you only have 10-50ng of the mRNA pool so only fentograms/attograms of the sequence of interest. I wonder if a PCR is sensitive enough to amplify this low amount of " specific" cDNA but you can always try it offcourse.
In the manual Promega Improm II RT they use as a control a minimum of 10ng of specific cDNA (page 6) if I understand it right.
hope this helps
Thanks a lot Phillip for your info. Acutally I just re-extracted RNA now and am doing RT. I still suspect that the problem was from the degraded RNA, since the RNA that I am interested in is quite rare in the pool. I will do the PCR and run a gel to check if there is some luck assigned to me from above today and let you know the results. Cheers man!