relative quantitation - validation experiment - (Aug/08/2006 )

Hi,

I need help with relative quantitation using ddCt method - with the validation experiment. I am using ABI 7500.

- Is it possible to decrease the reaction volume from 50 to 20 ul (for 96 well plate) ? When reducing the reaction volume to 20 ul do you also reduce the amount of cDNA added to the reaction?

- When validating is it necessary to run the endogenous control gene with all prepared dilutions?

It seems that when I want to run 2 endogenous control genes plus a target gene - for one sample chosen and 6 dilution and three replicates for each dilution - I will have 54 reactions - that seems quite a lot to me, but maybe this is the only option or is it possible to run the samples in duplicates?

Unfortunately no one in our lab has any experience with this. I´d appreciate any helpful comment.
Thanks

Mo

- Is it possible to decrease the reaction volume from 50 to 20 ul (for 96 well plate) ? When reducing the reaction volume to 20 ul do you also reduce the amount of cDNA added to the reaction?

I always use 20 ul instead of 50 ul reactions. You have to change that value in the software before you start the RT-PCR. I think you can reduce the amount of cDNA and reagents accordingly to the volume.

- When validating is it necessary to run the endogenous control gene with all prepared dilutions?

I would do. It is necessary to normalize your samples.

It seems that when I want to run 2 endogenous control genes plus a target gene - for one sample chosen and 6 dilution and three replicates for each dilution - I will have 54 reactions - that seems quite a lot to me, but maybe this is the only option or is it possible to run the samples in duplicates?

54 seems alright. Sometimes, I prepare only duplicates, but if you get a high standard deviation you won`t know which value you can trust. So, using triplets is better.