data analysis for RT_PCR - (May/28/2012 )

Hello all,
I wanted some help in my real time pcr data analysis. I want to study expression levels of a gene, say gene X on a 24 hour scale. i.e. I want to study how expression levels on gene x varies across a day. I use a ribosomal protein gene ( house keeping gene) as my control. So if i have to calculate the fold increase in my gene X using comparative Ct method , given that i have Ct values for gene X and endogenous control can i use equation fold increase = 2^ (Ct control-Ct gene X)? Because technically comparative Ct methods uses 2^( (Ct control-test gene)in test/treated sample- (Ct control-test) in control/untreated sample). This applies for studies where you have a treated and untreated sample which is not the same in my case. All i have is a single tissue sample and i want to know the expression level of gene x relative to endogenous control.
Thanks

In time-course studies, expression at zero timepoint is usually taken as control or "untreated" and the rest of samples as "treated". Then you use standard equation.

Expression of your housekeeping gene can change over time. I would use at least two independent housekeeping genes in a timecourse experiment (eg. Gapdh and Bactin), at least until you establish that your housekeeping gene is consistent.

Trof on Tue May 29 10:53:22 2012 said:

hmmm... i have not come across such method of analysis at least not in papers from my field... I think i will try that as well... thanks a lot!!!!

biznatch on Tue May 29 14:14:31 2012 said:

well yes I do agree with that but i presume not as much as the genes i study... and more over normalizing it with the housekeeping gene should take care of that error right!!! I use rp49 and beta actin as well and atleast till now i have not seen much variation across a day unless the cells are subjected to some sort of stress..