Calculation of gene expression 2^-ΔΔCt.

Question

[eugene]

Hello! I have a question related to the evaluation of gene expression.

"2^-ΔΔCt" should be calculated for each sample or should the average value be used?

Method 1 - The average value of "ΔΔCt" for 5 samples and from this average value "2^-ΔΔCt" is calculated.

Method 2 - No average values for 5 samples. "ΔΔCt" is calculated for each of the 5 samples and "2^-ΔΔCt" is calculated for each of the 5 samples.

Answer 1

[Kevin Blighe]

Hi,

I believe you messaged me on ResearchGate. You have followed my post, here: How to report and plot qPCR data - delta delta Ct

You already have 3 replicates per Animal and condition (Control Treatment); so, I think that you should be calculating the 2^-ΔΔCt separately per animal. If you wish, after this, you then then report the average 2^-ΔΔCt across all animals.

Kevin

[eugene]

Hello! It was me who wrote to you on the ResearchGate website. The only thing that bothers me about the 2^-ΔΔCt separate calculation approach is that the animals are not related to each other in any way. I mean, animal 26 is not a match for animal 99, etc.

[Kevin Blighe]

I think that the assumptions are still okay. Normally, our 'control' would just be 'healthy' human genomic DNA that is purchased by the lab; however, in your case, you have control samples. The Animals do not have to match across the Control and Treatment groups.

[eugene]

Thank you!