Individual or Combined samples - Run each sample separately-or combine and run once (Oct/29/2008 )

Hi,

I have a question in regards to the best way to set up a Real time PCR experiment. I want to compare the gene expression between two groups; Control and Treated for numerous Genes (up to 20).

I plan to use the LinReg method to calculate the Efficiency, and the REST (Pfaffl) method to determine the relative expression.

My question is therefore,

Is it better to run a Real time on each sample,

1) Control vs Treated 1, Control vs Treated 2, Control vs Treated 3 etc..Calculate the mean for Treatment and then determine if there is a significant up or down regulation, or

2) Combine Treated 1 + Treated 2 + Treated 3. Perform a Real time PCR Control vs Treated, and see if there is a significan up or dow regulation between these two groups?

Any advice appreciated.
Thanks,
Brett

-Brett14-

QUOTE (Brett14 @ Oct 29 2008, 09:57 PM)

It really depends on your experimental setup.

If you are planning to do stats then I would recomment the following:

1. In each run have a positive control, and a no template control
2. have 1 or 2 callibrators, samples with high expression of your sample (lower expression gives more varied intra and inter run variation), and use the average of them as your 'control' in the Pfaffl equation
3. use 2+ housekeeping genes as a reference

Then you have two series of numbers to do easy stats.

-maset-