Folding change by 2^DDCT method

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Sep 26, 2017

Dear Colleages,

I am doing a set of experiments to investigate if a gene knockout influences Virus replication. Briefly, we infected wild type cells and siRNA transfected cells and we achieved more than 90% of gene silencing. Now I want to quantify viral RNA inside the cells (not the Virus outside the cell). So that we have two Groups: Wild-Type and siRNA silenced. 
I ran Real Time PCR for my target gene as well as Beta Actin as a reference gene. When I Analyse the data using the 2^(ddCT) (meaning 2^(ct ref-ct target)) I get really strange values. 
Simply my question is: how to estimate how many times my gene is downregulated in comparison to WT?
For example , the wild type gives values of 0,0358 and the siRNA silenced cell 0,0019.

Could anyone help me with kind of analysis?

Thank a lot




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Roberto Rosati 6 months ago

Wait a sec, 2^(ddCT) is not 2^(ct ref-ct target). It is 2^[(ct ref-ct target)wt - (ct ref-ct target)si].

Go to the profile of 6 months ago

Hi Thanks for your reply.

So my current problem is: I have two cell lines: one is the WT and the other is the siRNA treated. Then I infected those cells with a virus. I want to investigate whether this silenced gene influences the virus replication. 
Since this gene is not normally expressed in the cells I have no control as this gene is only expressed when the cells are infected.
As I mentioned, I have two populations: one WT and one siRNA cells. In this case, both were infected with a virus and then I analysed the expression of a viral gene. What I got based on CT values is that the WT the expression of the viral gene is 20 CT and in the siRNA cells the expression is 35. So in fact there is an influence of the silenced gene on virus replication.
How should this be expressed as fold change using the 2^ddct formula since this gene is not normally expressed in the cells and I have no control (beta actin as a housekeeping gene is used in all the runs)

Many thanks


Go to the profile of Roberto Rosati
Roberto Rosati 6 months ago

Yes I understand, but what you need is a calibrator. Your calibrator, in my opinion, is the wild type cell line infected with the virus.

So let's say that the wild-type cell line infected with the virus had:
Beta-actin Ct = 24 (BAwt = 24)
Viral gene Ct = 20 (VGwt = 20)

And the siRNA-treated, virus-infected cell line had:
Beta-actin Ct = 23 (BAsi = 23)
Viral gene Ct = 35 (VGsi = 35)

Your calculations are:

a) dCt = VG-BA

dCt(wt) = 20-24 = -4
dCt(si) = 35-23 = 12

b) ddCt = dCt(wt) - dCt(si)
ddCt = -4 -12 = -16

2^ddCt = 2^-16 = 0,000015

That is, with these numbers you'd have a reduction of about 65,000x in virus target RNA compared to the infected, wt cell line.