1. What is qPCR Efficiency?

qPCR efficiency (E) measures how effectively template DNA is amplified during each PCR cycle. An ideal efficiency of 100% means the DNA quantity doubles with each cycle. The Thermo Fisher formula calculates efficiency from the slope of the standard curve.

2. How Does the Calculator Work?

The calculator uses the Thermo Fisher efficiency equation:

Where:

• \( E \) — PCR efficiency (dimensionless, often expressed as percentage)
• \( slope \) — Slope of Ct vs log(dilution) standard curve

Explanation: The slope of the standard curve relates to PCR efficiency. A slope of -3.32 indicates 100% efficiency (perfect doubling each cycle).

3. Importance of qPCR Efficiency

Details: Knowing your qPCR efficiency is crucial for accurate quantification, especially when using relative quantification methods. Efficiency between 90-110% is generally acceptable.

4. Using the Calculator

Tips: Enter the slope value from your standard curve plot (Ct values vs log template quantity). The slope is typically negative (-2.5 to -3.8).

5. Frequently Asked Questions (FAQ)

Q1: What is an ideal qPCR efficiency?
A: 100% efficiency (slope = -3.32) is ideal. 90-110% efficiency (slope -3.1 to -3.6) is generally acceptable.

Q2: Why is my efficiency below 90%?
A: Low efficiency may indicate PCR inhibition, poor primer design, suboptimal reaction conditions, or template degradation.

Q3: What if my efficiency is above 110%?
A: High efficiency may indicate primer-dimer formation, contamination, or inaccurate standard curve dilution series.

Q4: How often should I check qPCR efficiency?
A: Efficiency should be validated for each primer pair and checked periodically, especially with new reagent lots.

Q5: Can I use this with SYBR Green or TaqMan assays?
A: Yes, this efficiency calculation applies to both detection chemistries when using standard curves.