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RT-PCR calibrator

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#1 Bioscientist



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Posted 20 July 2011 - 10:51 PM

Hi guys

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.


#2 Trof


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Posted 21 July 2011 - 09:11 AM

Calibrator is just something you choose to compare to. If you have various tissues and no obvious calibrator (wt vs diseased, untreated vs treated,..) and just want to make a graph of them and don't have anything special to compare too, just pick one, maybe the one with more literature on it (like, many studied the expression of this gene in brain, so make brain tissue the calibrator) or the most abundant. It's required to be possitive for all genes (or use different calibrators for each gene that are positive).

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).

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#3 Bioscientist



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Posted 25 July 2011 - 10:05 AM

Thanks a lot Trof for the help. It cleared a lot of stuff.

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