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Confused about my CT values


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#1 Kongen

Kongen

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Posted 20 January 2014 - 01:11 PM

I just performed a qPCR experiment, but im a bit confused about the use of the Delta delta Ct method.

 

I have following results:

Normal conditions: 16.6 and 17.6 (Target gene)

Stress conditions: 18.24 and 17.3 (Target gene)

 

 

Normal conditions: 20.55 and 20.89 (Ref gene)

Stress conditions: 19.9 and 20.26 (Ref gene)

 

So initial what i want to do it see if the expression of the stress gene pr ref gene is higher in stress conditions than in normal condition.

Which by looking at the numbers should be, since the difference in Ct values for the samples are more closely related right?

 

However when I do the delta delta calculation (I use the average of the dublicates)

 

17.1 - 20.7 = -3.6

 

17.7-20.08 = -2.38

 

2^-(-2.38-(-3.6)) = 0.35

 

So as far as I understand the delta delta Ct this means that the expression in stress genes is 35% lower than in normal conditions right? Which I dont think really understand according to when i just look at the numbers. Is it because it uses total expression? The reason why I dont want this is that my organism may die under the more stress full conditions so the total expression might be lower.



#2 SusieQ

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Posted 21 January 2014 - 11:22 PM

As far as I know, the delta delta Ct isn't expressed in a percentage but in a fold change in expression. So 0.35 is not a percentage but a change in expression of your gene of interest in comparison to your reference gene.






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