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data analysis for RT_PCR

RT-PCR qPCR

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#1 nkl

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Posted 27 May 2012 - 11:58 PM

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.


#2 Trof

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Posted 29 May 2012 - 02:53 AM

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.

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#3 biznatch

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Posted 29 May 2012 - 06:14 AM

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.


#4 nkl

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Posted 29 May 2012 - 10:22 AM

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!!!!

#5 nkl

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Posted 29 May 2012 - 10:25 AM

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..





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