Selection of appropriate reference genes for quantitative real-time reverse transcription PCR in Betula platyphylla under salt and osmotic stress conditions

Abstract

Selecting appropriate reference genes is vital to normalize gene expression analysis in birch (Betula platyphylla) under different abiotic stress conditions using quantitative realtime reverse transcription PCR (qRT-PCR). In this study, 11 candidate birch reference genes (ACT, TUA, TUB, TEF, 18S rRNA, EF1α, GAPDH, UBC, YLS8, SAND, and CDPK) were selected to evaluate the stability of their expression in different tissues and under different abiotic stress conditions. Three statistical algorithms (GeNorm, NormFinder, and BestKeeper) were used to analyze the stability of the 11 candidate reference genes to identify the most appropriate one. The results indicated that EF-1α was the most stable reference gene in different birch tissues, ACT was the most stable reference gene for normal conditions, ACT and TEF were the most stable reference genes for salt stress treatment, TUB was the most stable reference gene for osmotic stress treatment, and ACT was the most appropriate choice in all samples of birch. In conclusion, the most appropriate reference genes varied among different experimental conditions. However, in this study, ACT was the optimum reference gene in all experimental groups, except in the different tissues group. GAPDH was the least stable candidate reference gene in all experimental conditions. In addition, three stress-induced genes (BpGRAS1, BpGRAS16, and BpGRAS19) were chosen to verify the stability of the selected reference genes in different tissues and under salt stress. This study laid the foundation for the selection of appropriate reference gene(s) for future gene expression pattern studies in birch.

Selection and Validation of Appropriate Reference Genes for Real-Time Quantitative PCR Analysis in Needles of Larix olgensis under Abiotic Stresses

“…No previous research has shown that TIP41 is the most stable RG under heat stress. ACT2 is a traditional housekeeping gene widely used in many species, such as Betula platyphylla under salt stress and Panicum virgatum L. leaves and roots [58,59]. In this study, ACT2 was an appropriate RG under heat stress, consistent with results from P. praeruptorum under heat stress [54].…”

Identification of Ginger (Zingiber officinale Roscoe) Reference Genes for Gene Expression Analysis

“…) have always been very popular. However, these genes have defects, and their expression varies greatly in different tissues, different developmental stages, and different experimental conditions (Chapman and Waldenström, 2015;Li et al, 2019). This is mainly related to the variability of reference genes in different environments and species.…”

Selection and validation of reference genes for quantitative real-time PCR of Quercus mongolica Fisch. ex Ledeb under abiotic stresses

“…Quantitative determination of expression levels of major gene through qRT-PCR is an important method to understand gene function and reveal the response mechanism of plants under adversities [ 21 , 22 ]. The accuracy of qRT-PCR is measured based on the choice of appropriate reference genes [ 31 ].…”