18S as a housekeeping gene for RT-PCR? getting wide variation
Posted 03 April 2009 - 01:02 PM
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.
Posted 03 April 2009 - 08:50 PM
Posted 03 April 2009 - 09:14 PM
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.