1. What is Copy Number in qPCR?

The copy number (CN) in quantitative PCR (qPCR) represents the relative quantity of a target DNA sequence compared to a reference. It's calculated using the ΔΔCt method which compares the cycle threshold (Ct) values between target and reference samples.

2. How Does the Calculator Work?

The calculator uses the standard ΔΔCt formula:

Where:

• \( CN \) — Copy number (relative quantity)
• \( \Delta\Delta Ct \) — Normalized Ct difference between target and reference

Explanation: The formula assumes 100% PCR efficiency (doubling every cycle). For different efficiencies, the formula would be \( (1 + E)^{-\Delta\Delta Ct} \) where E is efficiency.

3. Importance of Copy Number Calculation

Details: Copy number determination is essential for gene expression studies, viral load quantification, SNP analysis, and transgenic organism characterization.

4. Using the Calculator

Tips: Enter the ΔΔCt value obtained from your qPCR analysis. The value can be positive or negative depending on your experimental setup.

5. Frequently Asked Questions (FAQ)

Q1: What does a copy number of 1 mean?
A: A CN of 1 indicates the target sequence is present at the same quantity as your reference sample.

Q2: What if my ΔΔCt is negative?
A: Negative ΔΔCt values will result in CN > 1, indicating more copies than the reference. Positive ΔΔCt gives CN < 1.

Q3: How accurate is this calculation?
A: This assumes perfect PCR efficiency. For precise quantification, validate with standard curves and efficiency corrections.

Q4: Can I use this for absolute quantification?
A: No, this provides relative quantification. For absolute copy numbers, use standard curves with known quantities.

Q5: What about multiple reference genes?
A: For multiple reference genes, calculate the geometric mean of their Ct values before determining ΔΔCt.