Greetings to you all:
-I only got to test 4 ref. genes
-In spite of that, I obtain in geNorm M values lower than 0.5 and the best couple got a 0.217 M value.
-Then, I chosed 3 ref. genes since the variation of FN plot was less than 0.15 between V(3/4) but it was more if I just choosed two ref. genes V(2/3) .
I attached a file with M values obtained as well the FN plot.
I hope my details are understandable, I hope someone can shed a light in this matter.
thank you.
If you are working with homogeneous samples such as cells from cell culture, then choose genes with M < 0.5.
If working with heterogeneous samples such as tissue, then M < 1.0 is suitable.
In my opinion you do not have to use 2 genes because it has a lower M value when gene 3 is stable by itself ( M < 0.5).
I say this because it may be more convenient to use 1 ref gene rather than 2 if you have large number of samples. Double reference approach is usually necessary for tissue that is highly heterogeneous in expression such as tumor samples which often require multiple genes for normalization.
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