I would like to determine gene relative expression in real-time PCR using the comparative CT method (2-ΔΔCt), using three internal control instead of one gene.
More info on the issue is in the attached paper for example.
More info on the issue is in the attached paper for example.
You will need calculate the geometric mean of the Ct of these three genes. Then, you will apply the algorithm 2-ΔΔCt .
Alexander, Too many thanks appreciating you, just I got your answer.
Using two from three not optionally, we must use all of them, based on the experiment hypothesis.
I would agree with Alexander's method. Given your cirucmstance use qBase+ to identify your most stable genes, then find a geometric mean of your given three and follow with the 2-ΔΔCt method.
Is there any particular reason your hypothesis must use all 3 reference genes? Is there any way around it? Hoping to avoid scientific error if possible!
Ahmed many thanks
The reasons for using those three genes are based on our hypothesis, which aims to measure the gene expression using three different stop codon/ housekeeping, and as all of you know the translation termination occurs when one of the three stop codons, UAA, UGA and UAG, enters the ribosomal A-site, so that using all of them is obligatory.
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