real time RTqPCR: Combination of housekeeping genes

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real time RTqPCR: Combination of housekeeping genes

Postby Jacques » Feb 01 2017 5:03 pm

Hello everyone:
I'm working on RNA expression level of a given gene "A" (=my gene of interest) in cancer cells. I'm using Taqman probes.

I have a question about combining multiple housekeeping genes to improve the stability in RT PCR.
I have 40 samples. I have a calibrator-sample.
To calculate the ratio of my gene A compared to my housekeeping gene (GAPDH):
I used the following formula:
gapdh.gif


where:
A: gene A = gene of interest
Cal: calibrator
combines: combination of 3 genes
Ct: Theshold Cycle
ech: sample
E: efficiency


According to Normfinder, GAPDH's stability is insufficient, comparing it to two other housekeeping genes I also tested (ESD and POLR2A). To my surprise, ESD and POLR2A are not as stable as my gene A, which is not a housekeeping gene !!

A further investigation led me to this article dealing with "combining multiple housekeeping genes by using the geometric average of the Ct of each": https://www.ncbi.nlm.nih.gov/pmc/articles/PMC126239/
The aim of this article is to create a stable combinatory "housekeeping gene" when no single housekeeping gene can be found.

According to the article, the new Ct (Cycle Threshold) = Ct combines can be calculated as followed formula:
[See the next POST]

Thus, to calculate my gene A's Ratio for a given sample:
[See the 3rd POST]

So far, it seems to be ok... But how to calculate the efficicency of combination of these 3 housekeeping genes?
=> Is it also the geometric average of the efficiency of GAPDH, ESD and PLOR2A?

Thanks for your precious help!
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Last edited by Jacques on Feb 02 2017 9:02 am, edited 1 time in total.
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Re: real time RTqPCR: Combination of housekeeping genes

Postby Jacques » Feb 02 2017 7:14 am

Ref. post above:
moy geo.gif
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Re: real time RTqPCR: Combination of housekeeping genes

Postby Jacques » Feb 02 2017 7:17 am

3rd post (ref. 1st post):
combines.gif
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Re: real time RTqPCR: Combination of housekeeping genes

Postby Jacques » Mar 23 2017 10:21 am

I'm sorry. Just an Up to keep it alive...
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