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

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

Author information

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.

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

PMID:
26577777
DOI:
10.1007/978-1-4939-3115-6_2
[Indexed for MEDLINE]

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