Problem about RT-PCR for beta-actin - (Mar/30/2007 )

Dear all:
I try to amplify iNOS by using RT-PCR and I use beta-actin as internal control.I can get the iNOS band, but not beta-actin. When I did western blot I can get both iNOS and beta-actin band. So, can anyone tell me why I can't get beta-actin band from the RT-PCR.

Here is what I did: I extract total RNA using GStract RNA isolation kit and reverse transcript mRNA using oligo-dT20 primer to get the cDNA. Then I use iNOS and beta-actin as primers and cDNA as template to amplify iNOS and beta-actin DNA separately. The result is that I got the iNOS but not beta-actin.

Another question is that can I put the iNOS and beta-actin primer in the same tube to amply iNOS and beta-actin at the same time?

thanks

I wouldn't go to multiplex until you can get your singleplex working. and believe me, the beta-actin is in there, you just need to show it

It has to be one of 2 problems:
1. your primers are bad - re-design them
2. your conditions are not correct for the primers - re-design the conditions

how do your dissociation curves look?

Hi, thanks for your reply. I bought my primer from BIOSOURCE. I didn't design it by myself. And I did it under the instruction of the product. I think it is a very common primer for PCR. Do you have any other source of human beta-actin primer?
thanks