2 replies to this topic

[tank1917]

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!

[ElHo]

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

[tank1917]

ElHo, on 11 November 2010 - 03:59 AM, said:

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

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。