# negative values after GOI - reference gene ?? - (Jul/13/2011 )

Hi everyone,

in my real-time PCR I am using 40S as a reference gene which has a CT of about 25-27 and my GOIs have CT ranging from 23 - 30. This sometimes leads to negative values after the normalization (GOI - 40S). Is this a problem or can I still proceed with the delta delta CT analysis?

Thanks a lot!!

-Jo-Fish-

What is the exact formula you use to calculate relative normalised ratio?
There are some options how to calculate delta-delta Ct. Basically they differ in the +/- sign, so it may or may not be a problem.

-Trof-

Trof on Wed Jul 13 14:02:34 2011 said:

What is the exact formula you use to calculate relative normalised ratio?
There are some options how to calculate delta-delta Ct. Basically they differ in the +/- sign, so it may or may not be a problem.

Hey Trof,

I do following calculations: my formula is
Relative gene expression = 2^(-∆∆Ct)
With ∆∆Ct = (CT, Target – CT, Ref)Treatment - (CT, Target – CT, Ref)Control

This leads to some ∆∆Ct being positive and some being negative.

Thank you

-Jo-Fish-

The formula is OK.
It's normal to have negative delta-delta values, if you power 2 with them you will get below 1 for negative values and above 1 for positive. That means downregulation (below 1) or upregulation (above 1) of your samples compared to control (which is 1). 1 also equals 100%, so 0.5 would mean 50% of control.

-Trof-

Great!

Thank you very much!!

-Jo-Fish-