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Questions of Standards making in relative qRT-PCR


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#1 tank1917

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Posted 10 November 2010 - 10:33 AM

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

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 :wacko:

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

Thanks!

#2 ElHo

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Posted 11 November 2010 - 03:59 AM

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

#3 tank1917

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Posted 11 November 2010 - 05:37 AM

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。




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