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# negative values after GOI - reference gene ??

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### #1 Jo-Fish

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Posted 13 July 2011 - 01:17 AM

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!!

### #2 Trof

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Posted 13 July 2011 - 06:02 AM

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.
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### #3 Jo-Fish

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Posted 14 July 2011 - 12:23 AM

Trof, on 13 July 2011 - 06:02 AM, 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

### #4 Trof

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Posted 14 July 2011 - 07:50 AM

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.
Our country has a serious deficiency in lighthouses. I assume the main reason is that we have no sea.

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### #5 Jo-Fish

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Posted 14 July 2011 - 07:52 AM

Great!

Thank you very much!!