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

Heithem Ben Abdallah; Petra Bauer

doi:10.1007/978-1-4939-3115-6_2

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.

Authors

Heithem Ben Abdallah¹, Petra Bauer^{2

3}

Affiliations

¹ Institute of Botany, Heinrich-Heine University, Universitätstrasse 1, 40225, Düsseldorf, Germany.
² Institute of Botany, Heinrich-Heine University, Universitätstrasse 1, 40225, Düsseldorf, Germany. petra.bauer@uni-duesseldorf.de.
³ Center of Excellence in Plant Sciences (CEPLAS), Düsseldorf, Germany. petra.bauer@uni-duesseldorf.de.

PMID: 26577777
DOI: 10.1007/978-1-4939-3115-6_2

Abstract

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*