18S as a housekeeping gene for RT-PCR? getting wide variation - (Apr/03/2009 )
i am doing a few RT-PCR experiments and i am having some trouble with my housekeeping gene (18S). my ct values for my unknowns over the course of thee experiments are fairly consistent however my 18S is varying quite a bit. im not sure if 18S is known to be quite variable.
it is making the data quite difficult to interpret as my error bars are huge. if i had tight Ct values for 18S, things would look much better.
not sure if i am doing anything wrong since my sample Cts are all consistent and the only thing that is not is my 18S Ct values.
Have you run regular PCR using the primers for 18S rRNA or run the real-time PCR product to see if it gives you an unique band. You should also run a disassociation assay to see how the primers behave. 18s is supposed to be a better control than beta actin.
18S should be fairly constant. I did a test on samples treated with a harsh toxin and 18S more consistent than GAPDH and b-Actin. I guess it all depends what the unknowns are treated with.
have you ran a melt curve following the reactions to see if you get one peak?
Also have you optimized the 18S primers to make sure they are properly amplifying throughout each step.