qPCR ΔΔCt (Relative Quantification)
Calculate relative gene expression changes using the comparative Ct (ΔΔCt) method. Enter Ct values for target and reference genes in treated and control samples to compute fold change.
qPCR ΔΔCt (Relative Quantification)
advancedCalculate relative gene expression using the comparative Ct method
Formula
RQ = 2^(-ΔΔCt)
How It Works
The comparative Ct (ΔΔCt) method, also known as the Livak method, is the most common approach for analyzing relative gene expression from qPCR data.
Step 1: ΔCt (treated) = Ct(target, treated) − Ct(reference, treated)
Step 2: ΔCt (control) = Ct(target, control) − Ct(reference, control)
Step 3: ΔΔCt = ΔCt(treated) − ΔCt(control)
Step 4: Fold Change = 2^(−ΔΔCt)
Key assumptions: The reference gene must be stably expressed across conditions, and the amplification efficiency of both target and reference genes must be approximately equal (and near 100%).
Example
Investigating a gene's expression under drug treatment:
ΔCt(treated) = 25.0 − 20.0 = 5.00
ΔCt(control) = 28.0 − 21.0 = 7.00
ΔΔCt = 5.00 − 7.00 = −2.00
Fold Change = 2^(−(−2.00)) = 2^2 = 4.00
The target gene is 4-fold upregulated in the treated condition compared to control.
Frequently Asked Questions
What does a fold change of 1 mean?
A fold change of 1.0 means no change in expression between the treated and control conditions. Values > 1 indicate upregulation, and values between 0 and 1 indicate downregulation.
What is a good reference gene?
Common reference (housekeeping) genes include GAPDH, β-actin (ACTB), 18S rRNA, and HPRT1. The best reference gene should show stable Ct values across all experimental conditions.
What if my amplification efficiency is not 100%?
The ΔΔCt method assumes ~100% efficiency (doubling each cycle). If efficiency differs, use the efficiency-corrected method: Fold Change = (E_target)^(−ΔCt_target) / (E_ref)^(−ΔCt_ref).