(see attached diagram) I am doing a two-step qPCR. I incorporated different point mutations at the 3' end of my target RNA and am planning to measure the difference in RNA expression level using qPCR. For some reasons, I can't use primers located in the common upstream sequence (i.e. 5' region) between RNAs containing distinct 3' point mutations for reverse transcription but can only use the sequence downstream of the point mutations (i.e. farmost 3' region). However, the distance from my closest 3' point mutation to the farmost 3' end is only ~10 nt. So can I perform RT using an oligo containing only these 10 nt? For the real-time PCR step, I can use primers lied in the common 5' region.
You could use it, but make sure the the melting temperature is at least 50 deg C (Ideally 52-54), as reverse transcription is done at 42 deg C.
Usually the minimal length for primers is 15-18 oligos, so 10nt is on the short end, but if the melting temp is in the range I suggested you can still try, plus it sounds like you do not have many other options, unless you can design multiple primers which extend into the region of the mutations.
Hi Aleardo, thank you for the suggestion. I calculated the Tm of the 10nt oligo and it's only around 30 degree. So I don't think I can still use it. The thing is if I design different primers for different mutant RNAs, I'm worrying about the difference in the efficiency of the RT step. Does it matter to the final quantification?
Hi Leonard, I am not an expert in RT-PCR to quantify mRNA levels (I usually use reverse transcription for other purposes), but I think your concern is valid.
My understanding is that you have a mixture of different RNAs, and you want to quantitate their relative levels of expression. If that is correct you could use your 10nt oligo, which will decrease the efficiency of the reaction, but will not introduce biases towards any of the sequences under study (as they all have the 10nt region, and the binding will be random)
In order to increase the binding of your oligo first mix the template and the primer in presence of salt (to increase melting temp), heat at 70 deg C and let slowly cool down at room temp. After that add enzyme, nucleotide, buffer etc...and start the RT. I hope this helps. Let me know if you have other questions, and if I can I will help.
You can artificially increase the Tm of a short oligonucleotide by using modified nucleotides (e.g. LNA).
The ideal properties for the Gene specific primer in reverse transcription are: Length: 20 - 25 nt, Tm: 50 - 55 degrees and a GC percentage of 50 - 60 %.
For your case, the primer should not be that much close to the point mutation, you can always pick up a primer from intronic part that is a bit farther than the mutation (100 - 150 nt).
Hope that would help.
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