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Analyzing qrt PCR results

by gravenewworld
Tags: analyzing, results
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gravenewworld
#1
Nov10-12, 03:06 PM
P: 1,408
I have 5 different cell lines I'm testing and 2 different genes with GAPDH being used as my housekeeping gene which the mRNA for the 2 different genes are normalized to. People have told me that you can't compare the two different gene mRNA levels to each other, but can only compare the mRNA levels of the same gene across the different cell lines. I really don't understand why, is this true?
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Neghentropia
#2
Nov23-12, 02:08 PM
P: 18
of course you can compare them

what you can't do is to compare your genes of interest in all your different cells lines whithout referring them to the housekeeping.
gravenewworld
#3
Nov26-12, 05:05 PM
P: 1,408
You can? From what I was told you can't compare two different genes to each other due to the fact that you don't know how efficient each primer is for each gene (makes sense I guess). You can only compare the same gene across multiple cell lines after correcting with a housekeeping gene (in this case GAPDH). I'm doing relative quantitation and not generating any standard curves.

Neghentropia
#4
Nov27-12, 12:16 AM
P: 18
Analyzing qrt PCR results

oh, well, of course.
if you don't know the primers efficiencies you can't do any quantitation...

anyway if you compare genes on different cells lines you may want to use more than one single housekeeping


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