Primer Efficiency
Why efficiency matters
The DDCt method assumes 100% amplification efficiency for all primers. When efficiencies differ substantially (outside 90–110%), the Pfaffl method provides efficiency-corrected results.
AnnealIQ offers two ways to enter efficiency data: manual entry or standard curve calculation.
Manual entry
The efficiency card shows text inputs for each gene’s amplification efficiency (%). Validation thresholds appear inline with color coding: green (90–110%), amber (85–90% or 110–115%), red (below 85% or above 115%).
When efficiency is outside 90–110%, a recommendation message explains why Pfaffl is the better method choice.
Standard curve upload
Switch to the Standard Curve tab and upload a dilution series CSV. AnnealIQ plots the data points with a regression line and calculates: slope, R-squared, and efficiency.
The R-squared quality indicator uses the same color coding: green (> 0.99), amber (0.98–0.99), red (< 0.98).
Calculated efficiency is automatically applied to the selected gene and feeds into the Pfaffl analysis.