Advanced qPCR Analysis Pipeline: A Reproducible R Framework for Automated Gene Expression Quantification and Visualization

Abstract

This software repository enables the automated, high-throughput analysis of Quantitative Real-Time PCR (qPCR) data using the R programming language. Developed to enhance reproducibility in molecular oncology and genetics research, this pipeline streamlines the transition from raw Cycle Threshold (Ct) values to statistically validated publication-quality figures.

Methodology

The pipeline implements the comparative Ct method (Livak method, 2−ΔΔCt) to calculate relative gene expression fold changes normalized to a reference gene (e.g., GAPDH). It incorporates rigorous statistical testing, utilizing Welch’s t-test (accounting for unequal variances) to determine significance (p-values) and calculating 95% Confidence Intervals (CI) for precision estimation.

Key Capabilities:

Data Parsing: Flexible input handling for standard CSV formats.
Statistical Analysis: Automated computation of Fold Change, log2 Fold Change, Standard Error (SE), and significance levels.

Advanced Visualization:

Generation of high-resolution plots including Global Expression Profiles (Bar Plots), Treatment-Specific Volcano Plots, and Heatmaps with significance indicators.

Granular Reporting: 

Production of individual gene-level plots for detailed inspection.
Reproducibility The package includes a standardized demo dataset (simulating differential expression of 10 target genes under multiple treatment conditions) to facilitate immediate testing and verification of the workflow.

v1.1.0 - Advanced qPCR Analysis Pipeline

This release provides an updated and reproducible R-based pipeline for qPCR data analysis using the Livak method (2^-ΔΔCt).

Key updates

• Added standardized dummy qPCR dataset for reproducibility.
• Improved automated statistical analysis using Welch's t-test.
• Added fold change, log2 fold change, p-values, and 95% confidence intervals.
• Generated publication-quality visualizations including:
  • Global bar plots
  • Treatment-specific volcano plots
  • Expression heatmaps
  • Individual gene-level plots
• Updated documentation and citation metadata.
• Licensed under the MIT License.

Citation

Please cite the Zenodo archived version:

Noorollahi, H. (2026). Advanced qPCR Analysis Pipeline: A Reproducible R Framework for Automated Gene Expression Quantification and Visualization (v1.1.0). Zenodo. https://doi.org/10.5281/zenodo.20528637