how to calculate amount of cDNA using RT-PCR - (Nov/18/2014 )
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?
I think normalizing it to a ur standard that has a know copy number of gene would be a solution
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?