1. What is Delta Ct?

Delta Ct represents the difference in threshold cycles between a target gene and a reference gene in qPCR experiments. It's a crucial step in relative quantification of gene expression.

2. How Does the Calculator Work?

The calculator uses the Delta Ct equation:

Where:

• \( Ct_{target} \) — Threshold cycle for the target gene
• \( Ct_{reference} \) — Threshold cycle for the reference/housekeeping gene

Explanation: The equation normalizes the target gene expression to the reference gene, accounting for variations in sample quantity and quality.

3. Importance of Delta Ct Calculation

Details: Delta Ct is essential for comparing gene expression across samples and is the first step in calculating relative fold changes using the 2^(-ΔΔCt) method.

4. Using the Calculator

Tips: Enter Ct values for both target and reference genes. Values should be from the same sample run on the same plate under identical conditions.

5. Frequently Asked Questions (FAQ)

Q1: What is a good reference gene?
A: A stable housekeeping gene with consistent expression across all samples and conditions (e.g., GAPDH, β-actin, 18S rRNA).

Q2: What does a negative Delta Ct mean?
A: Negative Delta Ct indicates the target gene has lower Ct (higher expression) than the reference gene.

Q3: How precise should Ct values be?
A: Technical replicates should have Ct values within 0.5 cycles of each other for reliable results.

Q4: What's the next step after calculating Delta Ct?
A: Calculate Delta-Delta Ct (ΔΔCt) by comparing to a control sample, then use 2^(-ΔΔCt) to determine fold changes.

Q5: Can I compare Delta Ct values across different plates?
A: Only if the plates were run under identical conditions with proper inter-plate calibration controls.