qPCR analysis with error bars
(by pjie2 from cam.ac.uk)
Thu Dec 1 20:04:44 EST 2011
> 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.
Yes. The ±0.17 is the uncertainty in your reference (untreated) sample
though, not in the test sample. You should be plotting both in your
final figure, with the appropriate uncertainty on each.
So, in page 58 Step 5, after calculating ddCt, i.e. calibrating to the
untreated sample, for you have:
Drug A: -2.5 ±0.10
untreated: 0.0 ±0.17 <-- NOTE THAT THIS ZERO STILL HAS AN UNCERTAINTY
This is all clearly shown in Table 11, along with the figures for the
other two drug treatments. One place confusion may be coming in is that
for some reason Table 11 and the subsequent workings on pages 57-59 use
different figures for the s.d. for untreated/GAPDH. Specifically, in
table 11 this is given as ±0.09, whereas on page 57 (final line) they
use ±0.022. Carrying this difference forward is why page 59 line 3
gives the untreated dCt value as 6.86±0.17, compared to the value of
6.86±0.19 shown in table 11.
In all other respects the workings are accurate. The uncertainty in the
untreated sample stays with the untreated sample final value, the
uncertainty in the Drug A sample stays with the Drug A final value, and
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