Hi all, I am trying to design primers (I am using Primer3) for RT-qPCR on a bacterial gene (so the reaction will first do cDNA with reverse transcriptase and then qPCR). I am following a tutorial for the use of Primer3 since this is completely new for me. There is a part described in the tutorial that is not completely clear to me .It regards the option "excluded regions" and it says:
"This option is very helpful if a certain part of the sequence needs to be avoided. For instance, if oligodT primers are used to perform reverse transcription to create cDNA, the 5' end of the mRNA (if it is very long sequence) could not be represented in the cDNA as the oligodT primer may fall off before reaching it. In this case, it is necessary to avoid designing primers along the 5' end and instead target the 3' end of the sequence"..
Can you clarify to me better the meaning of this option?Can this section refer to the exclusion of introns from genes and other regions or does it refer to something else?I am uncertain if this applies to my case.. I will do reverse transcription from mRNA to cDNA befpre the qPCR..anyway, since I have bacterial genes and they have no introns, I think I don't need to exclude any region, do I?
Thank you so much
Hi Silvia,
the option that Primer3 gives you is for exclusion of target DNA sequences that you would like to avoid as Scott mentioned. The intron exclusion is also an good options once you design a primer.
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