6 replies to this topic

[gmo]

Hello everybody,

I’m currently planning an RT-PCR in order to clone my gene of interest. I’ve settled on a 2-step RT-PCR protocol using a specific primer at the retro-transcription (RT) step.
I’m now trying to design primers, one specific primer for the RT step and another pair for the PCR step having restriction site in 5’.
I understood that the reverse primer for the PCR step must contain the stop codon (as well as the forward must contain Kozac sequence and start site). However I get a little confused with the specific primer I want to design for the RT step. Indeed, to me that first primer must be very close – if not identical – to the reverse primer of the PCR step.
So my questions is: can I use the same reverse primer for RT AND PCR steps.
Is it something usual? And if so I suppose nothing prevents me from having a primer for RT step that possess a non-complementary RE site in 5’? Any good advice on this or closely related are welcome.

Finally, and I think this is linked somehow, I would like to know why the only type of priming possible in a one-Step RT-PCR is using a specific primer; I don’t see why hexon priming or oligodT priming would impeed the PCR step of the reaction, or is it due to another concern…

Thanks a lot for your future answers, can’t stand waiting to discuss that

Gmo

[pcrman]

If your purpose is to amplify the full length cDNA and clone it, all three types of primers (oligo dT, hexamer, and gene specific primer) should work for the RT reaction. They have their pros and cons.

Oligo dT primers offer specificity toward polyA mRNA, but if the mRNA is too long, you may not get full length cDNA (the reaction could not reach the very 5 end of the mRNA). Gene specific primers offer even more specificity toward your gene, but may have the same concern as oligo dT primers.

If you do want to use a gene specific primer for the RT reaction, you can use the same primer as the reverse primer in subsequent PCR reaction.  You can also design a GSP in the 3UTR for the RT reaction, then use use another reverse primer which is upstream of the GSP for the RT-PCR.

[gmo]

Ok thank you very much.
And I understand why hexamer priming is impossible in a one-Step RT-PCR, but eventually wouldn't oligodT priming be possible in this context where the tube is not opened from the beginning to the end of the reaction ?

[pcrman]

If you want to amplify the cDNA for cloning, why not two step RT-PCR? Once you convert your RNA into cDNA, you can use it for many PCRs.

[gmo]

Ok, but I just don't know if in a 2 step RT-PCR I need to purify the cDNA before the PCR? (in order to get rid of the RT pol and the hexamer/oligo dT primers to have specific amplification and a good PCR yield... )

[pcrman]

no, you don't need to purify the RT reaction, but you can dilute it, for instance, 1:4 using H2O.

[dandan]

hello all, i have met a similar problem, i used oligodt as primer to get the cdna and tried to amplify the target gene by pcr.
however, i dont see the band after running the gel, although the gapdh band is very strong. also RNA is not degraded at all.
so i was wondering if it is possible because the cdna is not fully transcribed, but how come the gapdh still works.
could you guys help. thanks.