Selection and Validation of Reference Genes for the qRT-PCR Assays of Populus ussuriensis Gene Expression under Abiotic Stresses and Related ABA Treatment

Ming Wei,Chenghao Li,Han Lu,Yingxi Chen,Jingli Yang,Xin Zhang,Mengqiu Zhang

doi:10.3390/f11040476

Abstract

Populus ussuriensis Kom. is one of the most important tree species for forest renewal in the eastern mountainous areas of Northeast China due to its fast growth, high yield, and significant commercial and ecological value. The selection of optimal reference genes for the normalization of qRT-PCR data is essential for the analysis of relative gene expression. In this study, fourteen genes were selected and assessed for their expression stability during abiotic stress (drought, high salinity, and cold stress) and after the treatment with the drought-related hormone ABA. Three algorithms were used, geNorm, NormFinder, and BestKeeper, and a comprehensive ranking of candidate reference genes was produced based on their output. The most appropriate reference genes were UBQ10 and RPL24 for drought and ABA treatment, UBQ10 and TUB3 for cold stress, and UBQ10 and 60S rRNA for high salinity. Overall, UBQ10 was the most stable reference gene for use as an internal control, whereas PP2A was the least stable. The expression of two target genes (P5CS2 and GI) was used to further verify that the selected reference genes were suitable for gene expression normalization. This work comprehensively assesses the stability of reference genes in Populus ussuriensis and identifies suitable reference genes for normalization during qRT-PCR analysis.

Highlights

Gene expression analysis is the basis of modern molecular biology and is widely used in genetic and developmental studies
PCR, and quantitative real-time PCR, all of which rely on normalization procedures to quantitatively compare multiple samples [1]. qRT-PCR has become a common technique for gene expression analysis because of its rapidity, high sensitivity, and quantitative accuracy [2,3]
A set of MIQE (Minimum Information for Publication of Quantitative Real-Time PCR Experiments) guidelines aimed at improving the reliability and repeatability of research using qRT-PCR, has been published, which refers to a suitable reference gene (RG) for normalization as being essential for obtaining reliable results [6]

Summary

Introduction

Gene expression analysis is the basis of modern molecular biology and is widely used in genetic and developmental studies. Gene expression can be analyzed by several RNA quantification methods, including Northern blots, RNase protection assays, semi-quantitative reverse-transcription. PCR, and quantitative real-time PCR (qRT-PCR), all of which rely on normalization procedures to quantitatively compare multiple samples [1]. QRT-PCR has become a common technique for gene expression analysis because of its rapidity, high sensitivity, and quantitative accuracy [2,3]. QRT-PCR is regarded as the only effective way to measure the expression of genes with low mRNA copy number [4]. A set of MIQE (Minimum Information for Publication of Quantitative Real-Time PCR Experiments) guidelines aimed at improving the reliability and repeatability of research using qRT-PCR, has been published, which refers to a suitable reference gene (RG) for normalization as being essential for obtaining reliable results [6].

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