Quantifying 35 transcripts in a single tube: model-based calibration of the GeXP multiplex RT-PCR assay

Area of Science:

• Molecular Biology
• Genomics
• Biotechnology

Background:

• Quantitative gene expression analysis is crucial in molecular life sciences.
• The Gene eXpression Profiling (GeXP) technology utilizes multiplex RT-PCR and capillary electrophoresis.
• GeXP enables quantification of at least 35 genes per reaction using a single dye.

Purpose of the Study:

• To establish a kinetic model for primer binding and PCR product formation.
• To provide a basis for creating and evaluating calibration curves for GeXP analysis.
• To validate the model and data processing workflow using Perl scripts.

Main Methods:

• Development of a kinetic model for PCR.
• Implementation of a data processing workflow with Perl scripts for calibration, evaluation, and quality control.
• Validation using RNA from differentiating Physarum polycephalum plasmodial cells.

Main Results:

• Demonstrated robustness and linearity in quantifying individual transcripts.
• Showcased accurate quantification even with variable relative abundance of co-amplified transcripts.
• Validated the kinetic model and data processing workflow.

Conclusions:

• GeXP analysis is a robust, sensitive, and precise method.
• It is suitable for quantifying tens to hundreds of genes in numerous samples.
• The method provides reliable gene expression profiling data.