Enter replicate Ct values separated by commas. This calculator supports 2^-ΔΔCt and efficiency-corrected Pfaffl analysis.
| Sample Group | Target Ct Replicates | Reference Ct Replicates | Target Efficiency | Reference Efficiency |
|---|---|---|---|---|
| Control | 24.6, 24.8, 24.7 | 19.2, 19.1, 19.3 | 2.00 | 2.00 |
| Treated | 22.1, 22.0, 22.2 | 19.0, 19.1, 19.0 | 2.00 | 2.00 |
Mean Ct = Sum of replicate Ct values ÷ Number of replicates.
Sample SD = √[ Σ(Ct − Mean Ct)² ÷ (n − 1) ].
Coefficient of Variation = (SD ÷ Mean Ct) × 100.
ΔCt = Mean Target Ct − Mean Reference Ct.
ΔΔCt = ΔCt Sample − ΔCt Control.
Relative Expression by 2^-ΔΔCt = 2−ΔΔCt.
Pfaffl Ratio = (Etarget)Ct control − Ct sample ÷ (Eref)Ref control − Ref sample.
Log2 Fold Change = log2(Relative Expression).
Percent Change = (Relative Expression − 1) × 100.
- Enter a target gene and a stable reference gene.
- Type replicate Ct values for control and test groups.
- Provide amplification efficiencies if known from validation runs.
- Select Pfaffl mode for efficiency correction or classic 2^-ΔΔCt mode.
- Press Calculate Expression to display results above the form.
- Review mean Ct, variability, fold change, and regulation direction.
- Use the export buttons to download CSV or PDF summaries.
1. What does this calculator measure?
It estimates relative gene expression between a control and a test sample using replicate Ct values, normalization against a reference gene, and optional efficiency correction.
2. When should I use Pfaffl instead of 2^-ΔΔCt?
Use Pfaffl when target and reference amplification efficiencies differ or when assay validation provides measured efficiencies. It improves accuracy over the simpler equal-efficiency assumption.
3. Why are replicate statistics important?
Replicate mean, SD, and CV help judge technical repeatability. Large variation may indicate pipetting issues, assay instability, or poor reaction consistency.
4. What Ct values should I enter?
Enter numeric Ct values from the same assay setup, separated by commas. Keep target and reference measurements matched to the correct sample group.
5. What does a relative expression above one mean?
A value above one means the target gene shows higher normalized expression in the test sample than in the control sample.
6. What does log2 fold change tell me?
It transforms the expression ratio into a symmetric scale. Positive values indicate upregulation, negative values indicate downregulation, and zero indicates no change.
7. Can I use more than three replicates?
Yes. The parser accepts any reasonable number of comma-separated numeric Ct values, then calculates summary statistics from all valid replicates.
8. Does the PDF export require external libraries?
No. This version builds a lightweight PDF directly in the script, making deployment simpler for single-file use cases.