How to do gen expression analysis with two endogenous control?

27 February 2025 0 2K Report

In my study, I am working with five different cell lines, two of which share the same genetic features and act as endogenous controls. My goal is to analyze gene expression data using the ΔΔCt method in qPCR.

Is it valid to take the average Ct value of these two control cell lines as a single endogenous control for ΔΔCt calculations?

Each of my control cell lines has three different passages, and for each passage, I have three technical replicates of qPCR data. What would be the correct approach to calculate the mean Ct value for these two cell lines? Should I first average the technical replicates per passage, then average across passages, and finally take the mean of both cell lines? Or is there a better statistical approach?

I appreciate any insights or recommendations

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