qPCR normalization of reference gene - (Mar/18/2012 )
I used SYBR green for my qPCR and GAPDH as the reference gene. I diluted all my cDNA for target gene primers in 1:6 ratio, and then I diluted another set of cDNA in 1:8 ratio for GAPDH(because the first one that I did with 1:6 is almost below Ct=15). How do I normalize the Ct values when analyzing the relative abundance?
Thank you so much!
This is my answer, but wait for other people on this forum to answer too:
if this is only for relative comparision then just calculate the delta Ct as always. unless you want to do quantification and draw standard curve? then to me it is risky to calculate, because there is a high chance of error. However, you can try reverse ratio. I mean for example if your Ct is 15 for a 1/8 sample, and you want to know the possible Ct for 1/6 you can try x=15*0.125/0.16=11.71. do this for any GAPDH sample.
I would recommend having same ratio cDNA for next experiment.
I used RotoGene software. However I don't remember if the software allowed adding the standard curve manually?!
see this handbook too: