TY  - JOUR
TI  - Efficiency-Corrected Relative Quantification of qPCR Data Using LinRegPCR and a Spreadsheet-Based Workflow
AU  - Müller, Louis Arnould
AU  - Tiret, Laurent 
VL  - 16
IS  - 13
PY  - 2026
DA  - 2026/07/05
SP  - e5729
C1  - Bio-protocol 2026;16:e5729
DO  - 10.21769/BioProtoc.5729
UR  - https://doi.org/10.21769/BioProtoc.5729
AB  - Quantitative real-time PCR (qPCR) is widely used for the quantitative assessment of relative transcript abundance in biological and medical research. Rigorous interpretation of qPCR data requires appropriate correction and normalization workflows that account for both technical variability and experimental heterogeneity. Regarding the correction step, the most used qPCR analysis relies on the 2-ΔΔCq method, which assumes identical and optimal amplification efficiencies across assays. Alternative strategies estimate amplification efficiencies using standard curves generated from serial dilutions, but these approaches require additional experimental work and may introduce serious dilution-related bias. Here, we describe a spreadsheet-based computational protocol for the correction of relative quantification of qPCR data that integrates amplification efficiencies derived directly from raw amplification curves using LinRegPCR. Cq values and per-reaction efficiency estimates are combined to calculate efficiency-corrected target quantities. Correction is then followed by normalization using the geometric mean of two reference genes. The workflow enables calculation of relative abundance fold-changes without the need for standard curves and produces output tables suitable for downstream statistical analysis. This protocol provides a transparent, dilution-free method for efficiency-corrected qPCR data analysis that can be implemented using commonly available software, facilitating reproducible and Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE)-compliant reporting of qPCR results.
KW  - Reproducible research workflows
KW  - PCR data quality control
KW  - Reference gene validation
KW  - RT-qPCR normalization strategies
KW  - Biostatistical analysis of transcript abundance
JF  - Bio-protocol
SN  - 2331-8325
PB  - Bio-protocol LLC.
BIO101  - False