18S changes with cDNA dilution
Posted 08 June 2009 - 07:44 PM
I am using 18S and beta actin as HKG across three treatments. What I have found is that the Cts are constant across these treatment groups, indicating these genes are appropriate HKG when I dilute the cDNA at 1:50. HOWEVER when cDNA is diluted 1:75 there is a significant difference in the expression of both 18S and beta actin across treatments, but not within treatments. This is a dilemma, as it suggests that these genes are not appropriate HKG. Does anyone have an explanation? or a recommendation?
Posted 08 June 2009 - 11:10 PM
Posted 09 June 2009 - 01:01 PM
Thanks, I thought that might have been the case. I will now screen various HKG - but of course may not come up with one that is appropriate. I'm tempted to just publish delta Ct relative to input RNA but given the feedback I get when I suggest this in discussions with various people, I expect quite a bit of resistance from reviewers/editors. Have you had any experience with publishing relative to input RNA?