Hello everyone,
I was just trying to do an RT-PCR with specific oligos to select the correct strand that I want to study. I cannot distinguish between both strands. So, I have tested different RT mixes:
-Enzyme, oligodT, Random Hexamers and buffer.
-Enzyme, strand specific primers for a gene and buffer.
-Enzyme and buffer.
-OligodT, Random hexamers and Buffer.
All of this mixes, with enzyme, are working and I can detect all the genes that I am studying.
I have discarded DNA contamination because my RNA is DNAse treated and also, when I do not use the enzyme for the RT, it does not work.
I have also tried different RT temperatures (37ºC, 42ºC, 50ºC, 55ºC), but it results the same for all of them.
How can I avoid that "only enzyme" works for the RT-PCR, and ensure a specific RT-PCR for my region of interest?
Thanks in advance,
Alvaro del Real
Hello Jack,
Until I clear my RT problem, I am using Taqman probes with the Normal mix RT (Enzyme, Hexamers, OligodT and buffer) and also, with the RT only enzime (enzyme and buffer). In both of them I can detect GAPDH (housekeeping gene).
If I do not use an enzyme I have not amplification. And my NTC does not amplify either.
I have just discarded a contamination in my buffer or enzyme, by using other new tubes too.
My equipment is 7300 real time PCR system (Applied).
Any ideas?
Thank you very much
Alvaro del Real There is a very simple reason that this is happening: Reverse transcriptase is able to use RNA as a primer and will extend the 3'-OH of an RNA primer by addition of dNTPs to form DNA. So in a mixture of only RNA+Enzyme the RNA is actually able to self-prime to some extent and allow the RT enzyme to make cDNA. Tiny bits of RNA formed by degradation or even hairpin structures at the 3' end of RNAs can make enough priming sites for reverse transcription to occur. This process is inefficient and has poor/inconsistent coverage of the mRNA sequences so that is why we use oligo-dT or random hexamers in routine reverse transcription. The correct control for qPCR is a reverse transcription reaction WITHOUT enzyme. Looks like everything is working just as it should for you because as you said, in the absence of enzyme you get no product. A reaction containing enzyme but no primers IS NOT an appropriate control for qPCR as there will be some self-priming by the RNA. Looks like you're doing everything right, just leave out the reaction with enzyme and without primer.
Best of luck!
Thank you Zachery R Belak . That totally answers my doubts and removes me from doing more tests. Now, my real problem is that I realized this fact by trying to perform a stranded reverse transcription to detect the antisense gene, without detecting the gene of the other strand. With this fact, I cannot amplify only my antisense gene without amplifying the sense gene. Any ideas? Thanks again for your answer.
You are correct that the cDNA that you make is antisense, however, if the application for the cDNA is PCR then it doesn't matter because in the first cycle of PCR the 5' primer will bind to the cDNA and make the second strand for you. Now you will have the sense strand and the antisense strand and PCR amplification will continue for both strands for the remainder of the PCR reaction. You do not need to do second strand synthesis to make the cDNA double stranded for PCR.
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