Molecular biology
How do you calculate qPCR fold change (ddCt)?
Livak 2^-ddCt with proper error propagation and an honest asymmetric band. The relationship used is fold = 2^(−ΔΔCt); band = 2^(−(ΔΔCt ± SD)).
Worked example
Every number below is computed by the calculator, not typed in
What you are given
- Treated target Ct (mean): 20
- Treated target Ct (SD): 0.3
- Treated reference Ct (mean): 18
- Treated reference Ct (SD): 0.2
- Control target Ct (mean): 22
- Control target Ct (SD): 0.25
- Control reference Ct (mean): 18
- Control reference Ct (SD): 0.15
Formula: fold = 2^(−ΔΔCt); band = 2^(−(ΔΔCt ± SD))
The working
- ΔCt treated / control:
Ct_target − Ct_ref = 2 / 4 - ΔΔCt:
ΔCt_treated − ΔCt_control = -2 ± 0.464 - Fold change:
2^(−ΔΔCt) = 4 (2.901 to 5.516)
Fold change 4x (up), 95% band 2.9 to 5.52.
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Try it yourself
Two problems, with the working revealed
Problem 1 qPCR fold change (ddCt)
Work out the result for qPCR fold change (ddCt).
- Treated target Ct (mean): 20.5
- Treated target Ct (SD): 0.5
- Treated reference Ct (mean): 18
- Treated reference Ct (SD): 0.25
- Control target Ct (mean): 22
- Control target Ct (SD): 0.15
- Control reference Ct (mean): 18.5
- Control reference Ct (SD): 0.1
Show the answer and the working
Fold change 2x (up), 95% band 1.33 to 3.01.
- ΔCt treated / control:
Ct_target − Ct_ref = 2.5 / 3.5 - ΔΔCt:
ΔCt_treated − ΔCt_control = -1 ± 0.587 - Fold change:
2^(−ΔΔCt) = 2 (1.331 to 3.005)
Open this in the calculator to change the numbers.
Problem 2 qPCR fold change (ddCt)
Work out the result for qPCR fold change (ddCt).
- Treated target Ct (mean): 23
- Treated target Ct (SD): 0.15
- Treated reference Ct (mean): 18
- Treated reference Ct (SD): 0.1
- Control target Ct (mean): 23
- Control target Ct (SD): 0.2
- Control reference Ct (mean): 19
- Control reference Ct (SD): 0.25
Show the answer and the working
Fold change 0.5x (down), 95% band 0.39 to 0.65.
- ΔCt treated / control:
Ct_target − Ct_ref = 5 / 4 - ΔΔCt:
ΔCt_treated − ΔCt_control = 1 ± 0.367 - Fold change:
2^(−ΔΔCt) = 0.5 (0.388 to 0.645)
Open this in the calculator to change the numbers.
Ten more problems on qPCR fold change (ddCt)
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