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

Authors
  • Marquardt, Pauline1, 1
  • Werthmann, Britta1, 2
  • Rätzel, Viktoria1, 3
  • Haas, Markus1
  • Marwan, Wolfgang1
  • 1 Otto-von-Guericke Universität, Magdeburg, Germany , Magdeburg (Germany)
  • 2 Present address: EKF-diagnostic GmbH, Ebendorfer Chaussee 3, Barleben, 39179, Germany , Barleben (Germany)
  • 3 Present address: Novartis Technical Operation – Solides, Barleben, Otto-von-Guericke Allee 1, Barleben, 39179, Germany , Barleben (Germany)
Type
Published Article
Journal
BMC Biotechnology
Publisher
Springer (Biomed Central Ltd.)
Publication Date
Apr 14, 2021
Volume
21
Issue
1
Identifiers
DOI: 10.1186/s12896-021-00689-4
Source
Springer Nature
Keywords
License
Green

Abstract

BackgroundQuantitative analysis of differential gene expression is of central importance in molecular life sciences. The Gene eXpression Profiling technology (GeXP) relies on multiplex RT-PCR and subsequent capillary electrophoretic separation of the amplification products and allows to quantify the transcripts of at least 35 genes with a single reaction and one dye.ResultsWe provide a kinetic model of primer binding and PCR product formation as the rational basis for taking and evaluating calibration curves. The calibration procedure and the model predictions were validated with the help of a purposefully designed data processing workflow supported by easy-to-use Perl scripts for calibration, data evaluation, and quality control. We further demonstrate the robustness and linearity of quantification of individual transcripts at variable relative abundance of other co-amplified transcripts in a complex mixture of RNAs isolated from differentiating Physarum polycephalum plasmodial cells.ConclusionsWe conclude that GeXP analysis is a robust, sensitive, and useful method when the transcripts of tens to few hundred genes are to be precisely quantified in a high number of samples.

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