Protocol Online logo
Top : Forum Archives: : Real-Time PCR

Genorm - Validation of housekeeping genes - (Jan/07/2008 )

Hi guys,

I am working in RT-PCR for some months and i am aware for the need of
proper validation of the internal control to be used in the
So, i have tested a 8 HSG in 15 samples (n=5). Genorm was applied to
this data and the best 2 genes present a M value of 0.412; nothing
wrong until now.

However, i also checked the variation of the NF. I got the following
V2/3 - 0.206;
V3/4 - 0.15;
V4/5 - 0.192;
V5/6 - 0.150;
V6/7 -0.222;
V7/8 - 0.208.
Looking for the value of V2/3 - 0.206 i would say
that 2 HSG for the normalization is OK, better 3; i cannot understand
how the NF is decreasing if more unstable genes are being included.
V5/6 - 0.15 is the lowest value, but this includes some genes poorly
ranked by genorm. I dont think that NF is affected by the absolute
number of HSG.

Any critics are welcome!



I have also used Genorm to find the best housekeeping genes for my samples. In the paper -Accurate normalization of real-time quantitative RT-PCR data by geometric avaraging of multiple internal control genes. Vandesompele J et al. Genome biol. 3:1-12, 2002- , they say: "Taking all this into consideration, we recommend the minimal use of the three most stable internal control genes for calculation of an RT-PCR normalization factor (NFn, n = 3), and stepwise inclusion of more control genes until the (n + 1)th gene has no significant contribution to the newly calculated normalization factor (NFn + 1). "
So, I have always used 3 housekeeping genes. I'm working with goat, so it takes a lot of time to design primers and test them, so we have made primers for 6 housekeeping genes and we took the 3 most stable ones (with M<1.5)