Questions of Standards making in relative qRT-PCR
Posted 10 November 2010 - 10:33 AM
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.
Posted 11 November 2010 - 03:59 AM
Posted 11 November 2010 - 05:37 AM
I isolate total RNA from tissue and reverse transcript total RNA into cDNA first stand by using Oligo dT.
What do you mean by cDNA synthesised from total RNA and complete cDNA?
And a PCR reaction used to amplify first stand into complete cDNA(two stand) with special primer.
My english is so poor……sorry。