3 replies to this topic

[p3t3r1]

Hey guys, I am doing RNA extraction, reverse transcribe it to cDNA and then try to do classic PCR and real-time PCR on some specific genes for quantification of gene expression. My supervisor told me to use different primers for real time PCR and classic PCR for these genes, it there a reason why other than to amplify different regions of the gene to use different primers?

[vladooo]

The reason your supervisor probably had on mind is that for the qPCR is more appropriate smaller PCR product (100 - 150 bp), while for classical PCR the product can be as long as your DNA polymerase and thermal program allows. The shorther PCR product for qPCR is necessary for highest PCR efficiency. Only when you have high PCR efficiency you can quantify precisely.

[bob1]

It all depends on the questions being asked - the qPCR is obviously for quantitation of gene response, but what is the standard PCR for?  Cloning? presence of the gene (qPCR will tell you that too)?

[p3t3r1]

vladooo, on Mar 31 2010, 02:07 PM, said:

The reason your supervisor probably had on mind is that for the qPCR is more appropriate smaller PCR product (100 - 150 bp), while for classical PCR the product can be as long as your DNA polymerase and thermal program allows. The shorther PCR product for qPCR is necessary for highest PCR efficiency. Only when you have high PCR efficiency you can quantify precisely.

Got it, thanks!