4 replies to this topic

[nkl]

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

Edited by nkl, 27 May 2012 - 11:59 PM.

[Trof]

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.

[biznatch]

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.

Edited by biznatch, 29 May 2012 - 06:14 AM.

[nkl]

Trof, on 29 May 2012 - 02:53 AM, said:

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.

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!!!!

[nkl]

biznatch, on 29 May 2012 - 06:14 AM, said:

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.

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..