Questions of Standards making in relative qRT-PCR - (Nov/10/2010 )

HI everyone:
I got some problems in my qRT-PCR again

I decide to use Pfaffl method to evaluate expression level of target gene（CaBP-9K） and reference gene（GAPDH）.But I don't konw how to prepare standards……Can I use cDNA first stand synthesised from total RNA for CaBP-9K and GAPDH standards?Or I must choose complete cDNA?But the cDNA first stand is the same for two genes……I am already confused

Sorry for my poor english and silly questions.Any advise is much appreciated.

Thanks!

What do you mean by cDNA synthesised from total RNA and complete cDNA?

ElHo on Thu Nov 11 11:59:01 2010 said:

I isolate total RNA from tissue and reverse transcript total RNA into cDNA first stand by using Oligo dT.
And a PCR reaction used to amplify first stand into complete cDNA(two stand) with special primer.
My english is so poor……sorry。