Identification and validation of suitable internal reference genes for SYBR-GREEN qRT-PCR studies during cucumber development

Abstract

SummaryQuantitative real-time RT-PCR (qRT-PCR) is currently the most popular method for measuring differential gene expression. However, selecting an inappropriate reference gene can lead to erroneous results by this approach. It is therefore important to identify the best reference gene(s) to use in each biological system before using qRT–PCR to investigate differential gene expression. In this paper, we evaluated the stability of expression of 13 different candidate reference genes in cucumber using the geNorm, NormFinder, and 'Stability Index' statistical algorithms. Twenty-six vegetative and 22 reproductive tissues or organs were sampled at different developmental stages. Based on our results, TUA, followed by ACT and UBI-ep, were found to be the most stably expressed genes, and were therefore suitable for normalisation purposes in vegetative tissue samples. ACT, ACT3, and TUA-1 exhibited high-stability expression and could be used as suitable reference genes to normalise gene expression in reproductive tissue samples. In addition, GAS1, which encodes galactinol synthase, displayed different levels of mRNA expression when normalised using stable or unstable reference genes. This identification and validation of suitable reference genes will facilitate future transcriptomic studies during cucumber development processes.