Understanding qPCR and gene expression changes - (Mar/31/2009 )
Myosin II on Thu Sep 17 08:16:32 2009 said:
What tells you if a gene is up or down regulated is your reference gene (also known as housekeeping gene). Likely you are aware about the use of genes like GAPDH, 18S and beta-actin as genes used to normalize the loading of RNA in experiments like Northern blots. qPCR uses genes like this for the same reason. The concept is that these housekeeping genes change very little, or not at all, in expression across your experiment so any change in the signal generated by these genes is due to things like differences in the amount of cDNA loaded in each reaction. By normalizing the signal generated by your genes of interest to the signal generated by a housekeeping gene then you can infer if your genes are up or down regulated.
I am just getting started with RT qPCR, but what I understand from my supervisor, many traditional housekeeping genes are actually not expressed as predictably as we like them to be and there is great variations among cell types and tissues. Therefore it should be stressed that a given tissue must be analyzed for proper normalization genes before doing your experiment, or there's a risk that you normalize to a gene that will lead to wrong interpretation of your data.
A former student of the lab did this work which explains the problem in more detail: Bonefeld BE, Elfving B, Wegener G: Reference genes for normalization: a study of rat brain tissue. Synapse 2008, 62:302-309