I want to know weather is it possible to perform the PCR using oligo dt primer and Gene specific primer using RNA as a template? if possible then how to calculate the annealing temperature to set in PCR
please help me.
Thanks and regards
Harshraj
you know at what temperature reverse transcriptase work ? I think in range of 37 - 42°C.. so you expect that they give you specific amplification???
I think you should go for :
cDna as template... first convert RNA to cDNA by using oligo dT or gene specific primer ...better use Gene specific primer ... then go with DNA polymerase like Taq or Pfu with gene specific and oligo dT primers..
yes some kits are available but the also first converts RNA to cDNA..
Hi There
Oligo dT routinely primes at between 65_70C (5_15min): This is your first step in RT using oligo dT primers in fact
Most gene primers typically prime between 55 and 65C
Whilst therefore in principle you could perform priming at 65C using a gfene specific forward primer and Oligo DT I agree with the above commentator:
Make cDNA using oligo dT and then amplify region/GOI using a gene specific forward and reverse primer. I suspect the latter might also be more specific; i.e. direct amplification using a GOI F primer and oligo dT is more likely to yield non specific bands
Harshraj Subhash,
You can use three types of primers for RT. These are the random primers, oligo dT and gene specific reverse primer (See link below). it is also good to know the length of the RNA and where about is located your gene of interest because it helps in getting good RT results. You should use Oligo dT if you know that the RNAs of your gene of interest has a poly A tail. Moreover using oligo dt with long RNA may not result in the 5' end not being transcribed if the enzyme falls off before reaching the 5' end.Using the random primers will be a better option in such case. The highest sensitivity you can achieve with RT is by using the Gene-specific reverse primer. In this case, all the RT activity is directed to the RNA of your gene of interest. Use the gene specific primers for your PCR reaction without leaving out the controls. Regarding the issue of temperature, you can follow the advice from the previous answers.
https://www.thermofisher.com/za/en/home/life-science/pcr/reverse-transcription/rna-priming-strategies.html
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