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how to calculate amount of cDNA using RT-PCR


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#1 molbio1234

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Posted 18 November 2014 - 01:41 PM

I am setting up reverse transcriptase qPCR on total RNA to measure the Ct values in the cDNA. I have relative standard curves in the plate as well, using control total RNA from a vendor and internal control RNA. Once the RT-qPCR measures the Ct values, how do I convert the Ct values to ng amount if this is a reverse transcriptase qPCR assay?



#2 Mohamed 1984

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Posted 18 November 2014 - 11:56 PM

I think normalizing it to a ur standard that has a know copy number of gene would be a solution 



#3 molbio1234

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Posted 20 November 2014 - 04:07 AM

I am using GAPDH as a standard gene. Please show me how I can normalize.

 

My RT qPCR reaction is 10uL volume. I'm adding 1uL of total RNA into it. If the total RNA of one of my relative standard points is 100ng/ul, then the cDNA is 10ng/ul if the qPCR was 100% efficient:

100ng/ul * 1uL = 100ng

100ng/10uL = 10ng/ul cDNA

 

If I don't know the total RNA concentration, how can I accurately quantify it by RTqPCR  using the relative standard curve? Do I use the y=mx+b formula?






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