semi-qRT-PCR - problem with housekeeping gene (Aug/16/2006 )
I'm trying to see the difference in upregulation of a gene upon different treatment using qRT-PCR.
I'm using one-step RT-PCR kit from Roche.
my problem is that when I amplify my gene of interest I can see the difference upon the different treatment but my housekeeping gene is saturated after 28 cycles of PCR !!!
what should I do? should I decrease the number of the cycles !!! or the RNA conc (since I'm using one step RT-PCR) I use 200 ng of RNA.
anyone has any idea ??
I think you should definitely try using less RNA. I normally use 25 ng RNA per reaction and have had no problems at all.
Give it a try! Best of luck