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How to do SEM and SD for qPCR in Excel?


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#1 Trof

Trof

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Posted 16 June 2011 - 05:50 AM

I will have a bit basic question, but I want to clear this problem once and for all.

I'm doing real-time expression studies and the raw output is a Ct value of replicate samples and their SD. I have one or more housekeeping genes to which I normalise my gene of interest. I can use relative quantification module to calculate the relative expression and SD, but sometimes I can't use it and though I can do normalisation with a few steps in Excel, the SD (or SE) is more dificult.

Lets say I have these values (three replicates):
Cts of housekeeping of the calibrator: 22.1, 21.9, 22.2 (mean 22.07)
Cts of housekeeping of the sample: 21.9, 21.8, 22.0 (mean 21.9)

Cts of gene of interest of the calibrator: 18.1, 18.3, 18.00 (mean 18.13)
Cts of gene of interest of the sample: 25.3, 25.6, 25.2 (mean 25.37)

There are many ways how to calculate the rel quant, but let's say I use delta-delta Ct, so normalised relative quantity of sample is = 0.006 (for calibrator it's of course 1)

I can calculate SD of the Cts, by the Excel function (it's better to use STDEV or STDEVP in this case? I would say it's not a part of population, but complete three measurements, so STDEVP should be used, but everyone uses STDEV). But I don't know how to calculate SD of the normalised relative quantity in Excel. Can I do it somehow from SD of the Cts? I've seen the formulas in papers, but those are gravely complex, so I would appreciate an Excel-based solution.

I got a spreadsheet from some other colleague, but that makes SD from the 2^Ct of each replicate and then using STDEV on them (she then calculates CV from that and finaly SE of normalized relative value, the SD in this case is a huge number, because it's used for 2 to power of a double-digit number so it's not a SD of the final result but of something else). I'm really not convinced this is the right method. Does anyone have a spreadsheet with some elegant calculation of SD and SE?

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