1. What is qPCR Efficiency?

qPCR efficiency (E) indicates how effectively the PCR reaction amplifies the target sequence. An ideal efficiency is 100%, meaning the product doubles each cycle. The Thermo Fisher model calculates efficiency from the standard curve slope.

2. How Does the Calculator Work?

The calculator uses the Thermo Fisher efficiency equation:

Where:

• \( E \) — PCR efficiency (converted to percentage)
• \( S \) — Slope of the standard curve

Explanation: The equation converts the standard curve slope into a measure of amplification efficiency. Perfect efficiency (100%) corresponds to a slope of -3.32.

3. Importance of Efficiency Calculation

Details: Knowing your qPCR efficiency is crucial for accurate quantification, especially when using relative quantification methods. Efficiency affects the calculation of fold changes in gene expression studies.

4. Using the Calculator

Tips: Enter the slope value from your standard curve. Typical values range from -3.0 to -3.6 (corresponding to efficiencies of 90-120%).

5. Frequently Asked Questions (FAQ)

Q1: What is an acceptable qPCR efficiency range?
A: 90-110% is generally acceptable, with 95-105% being ideal. Values outside this range may indicate problems with primers, template quality, or pipetting accuracy.

Q2: How do I get the slope value?
A: The slope is obtained from the standard curve plot in your qPCR analysis software, typically when running serial dilutions of your target.

Q3: Why is my efficiency above 100%?
A: Efficiencies >100% may indicate PCR inhibition in more concentrated samples, pipetting errors, or non-specific amplification.

Q4: What if my efficiency is below 90%?
A: Low efficiency suggests suboptimal reaction conditions. Consider checking primer design, template quality, or reaction components.

Q5: Can I use this for SYBR Green and probe-based assays?
A: Yes, this calculation applies to both detection chemistries, as long as you have a valid standard curve slope.