Hi!
I'm setting up an experiment to see the relative change in expression levels of some genes in M. smegmatis and I wonder what should be the appropriate reference genes for qPCR?
I found one - mysA, which is a sigma-factor, homolog of sigA in M. tuberculosis, and I need 1 or 2 more housekeeping genes with different levels of expression for a good set of controls.
Could you suggest any? If possible, with some citations=)
Thanks!
First of all you have to test the possible reference gen vs the gen of interest. If you have several genes of interest, you should test the reference gen vs each of them.
To verify the stability of the reference gen you will need to run different dilutions of your sample, in 5 magnitud orders at least. Once you have the Ct values of both genes (reference+interest), you have to plot Ct vs Log Conc. What you expect is to have a graph with 2 horizontal lines, parallels, that never touch each other. For each line you are also expected a slope = 0 or closest to 0. This is an indicative of stability, which means that your selected endogenous works for your particular type of sample when testing that particular gene of interest.
If the graph does not show the mentioned characteristics, then you should select another reference gen.
This publication may help: http://www.gene-quantification.net/livak-2001.pdf doi:10.1006/meth.2001.1262
Hi,
The best reference gene would be a gene that doesn't with your treatment (stable).
I would advice 18S as it is so abundant that it shouldn't change with any treatment.
Asif
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