Question: How to choose calibrator (housekeeping) genes for RT-qPCR normalisation
0
gravatar for peter pfand
3.8 years ago by
peter pfand80
Germany
peter pfand80 wrote:

Hi,

I am using geNorm and normFinder methods (implemented in NormqPCR R package) to find housekeeping genes from AP (life tech) cards, that stay stable across samples. The reason behind is  the (supposed) housekeeping genes do not behave as such, and they are highly variable across and even within samples. For instance U6 snRNA-001973 calibrator gene  may vary between 4 and 10 Cts within samples, for all of them.

Therefore, I get several stable housekeeping genes (with geNorm and NormFinder), but they show high Ct values (around 33-34), and I do not really know if I should trust them.

Furthermore, I get very low correlations or nothing at all between biological replicates, before and after normalisation with the computed housekeeping genes, which makes very hard for me to trust differential expression of different conditions, when the processed data is so unreliable.

I am a newbie with RTqPCR data analysis and do not know if it is possible to make magic with these apparent noisy data, and would really appreciate some feedback, comments or advice.

ADD COMMENTlink modified 3.6 years ago by karl.stamm3.6k • written 3.8 years ago by peter pfand80

Are you comparing across tissues or between experimental treatments?

ADD REPLYlink written 3.6 years ago by jotan1.2k
1
gravatar for karl.stamm
3.6 years ago by
karl.stamm3.6k
United States
karl.stamm3.6k wrote:

There's a pretty good investigation of the stability of housekeepers over here.

ADD COMMENTlink modified 15 months ago by RamRS25k • written 3.6 years ago by karl.stamm3.6k
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