Posted 20 July 2011 - 10:51 PM
I have a question regarding RT-PCR. I am comparing the expression profile of different genes in different tissues. I am including endogenous control genes in each plate. I am confused as what to use as calibrator as I am working with tissues from wild-type. Is it possible to determine the expresssion profile of these genes without including a calibrator using the delta delta ct method? How do I compare the expression of a gene in different samples using the endogenous controls only and no calibrator sample?
Thanks in advance.
Posted 21 July 2011 - 09:11 AM
Using a calibrator would make your quantity values look visibly better, like in this example:
sample A, Cts: control = 17 gene = 22, relative quantity 0.03125
sample B, Cts: control = 17 gene = 25, relative quantity 0.00390625
[relative quantity was calculated as 2 power to (Ct control - Ct gene)]
You can see the values are not very nice.
Now if you choose A as a calibrator, you have normalised relative quantity of sample A 1 and of sample B 0.125. And you can say in B there is only 12.5 % of A quantity (that is 8-fold decrease).
I never trust anything that can't be doubted.
Posted 25 July 2011 - 10:05 AM