Hi, I want to analyze my qPCR (gene expression, mechanical testet and not testet) results, and have to normalize them to my reference gene GAPDH. I don't have a standard curve.

I have looked at to methods, and can't decide if I should use one of them, or both.

1) is the 2^(delta delta Ct) which gives a fold between the testet or not normalized to GAPDH

2) is just dividing the sample Ct with GAPDH Ct and then compare the two results from testet and not testet samples.

What do you think?

Hi Amalie,

If I understand correctly your second option would only mean dividing one Ct over the other.

By doing this, for example with Ct values 30 and 25, you would get a fold induction/reduction of 30/25 or 25/30 resp.

However, this arithmetic does not respect the exponential nature of a PCR reaction where for each cycle the template product is duplicated/doubled*.

A reaction with a Ct value of 30 has 32 times less template than a reaction with a Ct value of 25. Since 2^(30-25) = 32 (or 2^-(25-30), actually).

Thus, you should go for option 1.

* Theoretically.

Each primer-set performs slightly different.

If you know your primer performance you can adjust your calculations.

Say 90% performance = 1.8x product increase for each cycle.

Then the math becomes 1.8^(ddct)

This would depend on how you would like to interpret the data

If it's just to show relative expression then delta ct= target gene expression- housekeeping gene expression would suffice. However, interpretation by may be difficult for those not familiar with the same.

Say you have a group that you would like to use as standard

Then 2 raised to-ddct would be an ideal way to express the values, where high values will refer to high expression and low like wise

Martin Huisman I dont know my primer performance, unfortunately. And I haven't had time for making a standard curve to test it. Will method 1 still be the best to go with?

Can I find the primer performance on the internet?