Hello everyone,

I am new to qPCR-based methods and trying to understand how the

monochrome multiplex quantitative PCR (MMqPCR) method, developed by

Cawthon (2009), calculates the T/S ratio for relative telomere length

measurement.

From what I gather, this method measures the telomere (T) and

single-copy gene (S) signals in the same well, but I am struggling to

understand how exactly the T/S ratio is calculated.

Could someone explain the step-by-step process, preferably with an

example? Specifically, I’d like to understand:

How the Ct values for T and S are used in the calculations?

Have they used the generally used delta ct method (not explicitly

mentioned in their paper)?

what is the formula used to compute the relative T/S ratio in a dataset?

How to normalize the values when comparing multiple samples?

A worked-out example with actual Ct values would be incredibly helpful!

Thanks in advance for your guidance.