1. What is ΔΔCt in qPCR?

The ΔΔCt method is a relative quantification strategy used in quantitative PCR (qPCR) to analyze gene expression changes between samples. It normalizes target gene expression to a reference gene and compares it to a control sample.

2. How Does the Calculator Work?

The calculator uses the ΔΔCt formula:

Where:

• \( Ct_{target} \) — Cycle threshold of target gene in test sample
• \( Ct_{ref} \) — Cycle threshold of reference gene in test sample
• \( Ct_{control} \) — Cycle threshold of target gene in control sample
• \( Ct_{ref\_control} \) — Cycle threshold of reference gene in control sample

Explanation: The formula first calculates ΔCt (target - reference) for both test and control samples, then subtracts the control ΔCt from the test ΔCt to get ΔΔCt.

3. Importance of ΔΔCt Calculation

Details: ΔΔCt is crucial for relative gene expression analysis. The fold change (2^-ΔΔCt) shows how much more or less the target gene is expressed in the test sample compared to the control.

4. Using the Calculator

Tips: Enter Ct values from your qPCR experiment. All values must be valid numbers. The calculator provides both ΔΔCt and fold change (2^-ΔΔCt).

5. Frequently Asked Questions (FAQ)

Q1: What is a good reference gene?
A: A stable housekeeping gene with consistent expression across samples (e.g., GAPDH, β-actin, 18S rRNA). Validate its stability in your experimental conditions.

Q2: What does a ΔΔCt of 0 mean?
A: A ΔΔCt of 0 means no difference in expression between test and control samples (fold change = 1).

Q3: What if my ΔΔCt is negative?
A: Negative ΔΔCt indicates higher expression in test sample compared to control (fold change > 1).

Q4: How many replicates should I use?
A: Minimum of 3 technical replicates per sample, with biological replicates for robust statistics.

Q5: What PCR efficiency is assumed?
A: The 2^-ΔΔCt method assumes 100% PCR efficiency. For different efficiencies, use efficiency-corrected calculations.