Quantitative Reverse Transcription-qPCR-Based Gene Expression Analysis in Plants

Methods Mol Biol. 2016;1363:9-24. doi: 10.1007/978-1-4939-3115-6_2.


The investigation of gene expression is an initial and essential step to understand the function of a gene in a physiological context. Reverse transcription-quantitative real-time PCR (RT-qPCR) assays are reproducible, quantitative, and fast. They can be adapted to study model and non-model plant species without the need to have whole genome or transcriptome sequence data available. Here, we provide a protocol for a reliable RT-qPCR assay, which can be easily adapted to any plant species of interest. We describe the design of the qPCR strategy and primer design, considerations for plant material generation, RNA preparation and cDNA synthesis, qPCR setup and run, and qPCR data analysis, interpretation, and final presentation.

Keywords: Cq value; Gene expression; Primer design; Reference gene; Reverse transcription; cDNA; qPCR.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Data Interpretation, Statistical
  • Gene Expression Profiling*
  • Gene Expression Regulation, Plant*
  • Plants / genetics*
  • Real-Time Polymerase Chain Reaction* / methods
  • Real-Time Polymerase Chain Reaction* / standards
  • Transcriptome*