qPCR Delta Delta Ct Calculator - Fold Change with Error
Ct values in, fold change out - with the standard deviation, and the right method picked for your amplicon efficiencies.
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The 2^-ddCt shortcut assumes both your amplicons double every cycle. When they do not - and a standard curve almost never says exactly 100% - the answer is wrong by a factor that compounds with every cycle of ddCt, so an efficiency of 1.9 against 2.0 is already a 1.6-fold error after ten cycles. This calculator picks the method from the efficiencies you give it rather than leaving that to you: Livak when both amplicons qualify, the Pfaffl efficiency-corrected ratio when they do not, and it reports what Livak would have said so you can see the size of the difference. Replicates give you a standard deviation on the fold change, computed on the log scale because a confidence interval that is symmetric in ddCt is not symmetric in fold change.
Treated / test condition. Replicates separated by commas.
Housekeeping gene, same sample.
Calibrator / untreated condition.
Housekeeping gene, control sample.
2.0 = 100%. From a standard curve, E = 10^(โ1/slope).
Leave both at 2.0 for the Livak 2^-ฮฮCt method.
ยฑ1 SD: 4.872ร to 5.718ร. The interval is symmetric in ฮฮCt, so it is not symmetric in fold change.
How to use the qPCR ddCt Calculator tool
- 1Paste the Ct values for your gene of interest in the treated sample - replicates separated by commas.
- 2Do the same for the housekeeping gene in that sample, and for both genes in your control sample.
- 3If you have measured amplification efficiencies from a standard curve, enter them as a per-cycle fold (2.0 = 100%).
- 4Read the fold change, its log2, and the +/- 1 SD interval when your replicates support one.
Frequently asked questions
What is the difference between the Livak and Pfaffl methods?
Livak's 2^-ddCt assumes both the target and the reference amplicon amplify with 100% efficiency, so each cycle exactly doubles the product. Pfaffl raises each amplicon to its own measured efficiency instead, which is what you need when a standard curve says they differ. This tool uses Livak only when both efficiencies are 2.0 and switches to Pfaffl otherwise, reporting the Livak answer alongside so the gap is visible.
How do I convert a standard-curve slope into an efficiency?
E = 10^(-1/slope), which gives the per-cycle fold - the number this tool wants. A slope of -3.32 gives E = 2.0, or 100% efficiency. Note that a percentage like 95% is not the value to type here: enter 1.95.
Why is the error interval on the fold change not symmetric?
Because the uncertainty is symmetric in ddCt, and fold change is 2 raised to the negative of it. Mapping both ends of the ddCt interval through that exponential gives an interval that is wider above than below, which is the honest shape. Reporting a single +/- figure on the fold change would be wrong on one side.
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