1. What is QPCR Efficiency?

QPCR (Quantitative Polymerase Chain Reaction) efficiency measures how effectively DNA is amplified during each PCR cycle. It is a critical parameter for accurate quantification of gene expression.

2. How Does the Calculator Work?

The calculator uses the QPCR efficiency equation:

Where:

• \( Slope \) — The slope from the standard curve plot of Ct vs. log starting quantity

Explanation: The equation calculates amplification efficiency based on the slope of the standard curve. An ideal efficiency is 1 (100% amplification per cycle).

3. Importance of Efficiency Calculation

Details: Accurate efficiency calculation is crucial for reliable quantification in qPCR experiments. It affects the accuracy of relative quantification and comparison between different genes or samples.

4. Using the Calculator

Tips: Enter the slope value obtained from your standard curve. The slope should be a negative value typically between -3.0 and -3.6 for optimal efficiency.

5. Frequently Asked Questions (FAQ)

Q1: What is the ideal qPCR efficiency?
A: The ideal efficiency is 1 (100%), which corresponds to a slope of -3.32. Efficiencies between 0.9-1.1 (90-110%) are generally acceptable.

Q2: Why is efficiency important in qPCR?
A: Efficiency affects the accuracy of quantification. Unequal efficiencies between target and reference genes can lead to errors in relative quantification.

Q3: How is the slope determined?
A: The slope is obtained from a standard curve plot of Ct values versus the logarithm of the starting template quantity.

Q4: What factors affect qPCR efficiency?
A: Primer design, template quality, reaction conditions, inhibitor presence, and instrument performance can all affect efficiency.

Q5: How often should efficiency be calculated?
A: Efficiency should be calculated for each primer pair and validated regularly, especially when changing reaction conditions or using new batches of reagents.