qPCR analysis with error bars

TR via methods%40net.bio.net (by taskan4 from gmail.com)
Fri Dec 2 04:39:56 EST 2011


> Have a look at this manual:
> http://www3.appliedbiosystems.com/cms/groups/mcb_support/documents/generaldocuments/cms_042380.pdf
>
> Step 6 (pag. 58) has the following title:
> Incorporating the standard deviation of the ΔΔCT values into the fold
> difference.
> _______________________________________________
>
> Yes, I've seen that.  Their example problem doesn't
> contain any biological replicates.    Taking biological variation
> through the calculations is what is confusing me.
>
> I also find their "step 5" to be very suspect.
>
> "The calculation of ΔΔCT involves subtraction of the ΔCT calibrator value. This is
> subtraction of an arbitrary constant, so the standard deviation of the ΔΔCT value is the
> same as the standard deviation of the ΔCT value.
>
> Therefore, ΔΔCT Drug Treatment A sample =
> ΔΔCT = 4.37±0.10 - 6.86±0.17 = -2.5±0.10"
>
>  6.86±0.17 sure doesn't look like an arbitrary constant.   It looks like a measured
> value with uncertainty that will affect the outcome of the calculation.
-------------------------------------------------------------

Well,
I am not statistician, we probably need one here. However, the
subtraction in step5, as I see it, serves only to give an arbitrary
value of 1 to the expression of your gene in the calibrator. Actually
you do not need to do it, the bars of relative expression for your
samples would look just the same, only that the scale in the axis
would be different: instead of "expression relative to the calibrator"
you would have "expression in arbitrary units": Imagine a set of
samples, from which you consider the sample A to be the calibrator:

If you do the subtraction, the relative expression could be:
A: 1
B: 2
C: 4
D: 8

If you do not do it the relative expression in arbitrary units would be:
A: 300
B: 600
C: 1200
D: 2400

The biological interpretation of the experiment would be the same in
both cases, since you do not know, in any of them, any absolute value
for the amount of the specific mRNA. So the standard deviation which
is relevant is the one that is used in the second case, that is, that
of ΔCT.

Hope this helps,
TR.



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