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i performed a real time PCR. I was looking for expression fold changes for 2 genes and i had two sample pools, one treated and the other not treated (for each gene). The problem is that my housekeeping gene (UBQ 30) got a fold change on the treatment. Therefore, what is the correct interpretation and data treatment?

i mean, is it correct to use each control and treatment i got for UBQ CT values to normalize the other two genes corresponding CT, as housekeeping genes are "by definition" expected not to change? (and that is why indeed they are used to normalize)

or should i rather make a mean of my whole UBQ CT-s and normalize all other genes CT values with that mean?

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In principle the housekeeping genes should be constant. But: Changes, that happen to the whole cell, can happen. So it is advisible to have more than one housekeeping gene if possible. –  Chris Mar 17 at 16:46

1 Answer 1

You should try another housekeeping gene like GAPDH since the very definition of these genes is that they shouldn't change with your treatment

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good advice - this is a list of even better ones...ncbi.nlm.nih.gov/pubmed/17394773 –  shigeta Mar 19 at 3:57
    
There is quite a list of genes that can be used - sometimes even GAPDH or Actin change. This needs to be verified for each experiment. –  Chris Mar 19 at 6:32
    
and how could i objetively discrimine if a given change for CT values is significable as to consider to change my housekeeping gene? –  Katz Mar 19 at 11:07
    
As Chris says you will have to verify that your housekeeping gene doesn't change between experiments. –  V_ix Mar 19 at 14:34

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