Identification and Validation of Reference Genes for Gene Expression Analysis in Schima superba.

Zhongyi Yang,Zhichun Zhou,Rui Zhang

doi:10.3390/genes12050732

Zhongyi Yang, Zhichun Zhou + Show 1 more

Open Access

https://doi.org/10.3390/genes12050732

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Journal: Genes	Publication Date: May 13, 2021
Citations: 31	License type: CC BY 4.0

Affiliation: Chinese Academy of Forestry, Nanjing Forestry University

Abstract

Real-time quantitative PCR (RT-qPCR) is a reliable and high-throughput technique for gene expression studies, but its accuracy depends on the expression stability of reference genes. Schima superba is a fast-growing timber species with strong resistance. However, thus far, reliable reference gene identifications have not been reported in S. superba. In this study, 19 candidate reference genes were selected and evaluated for their expression stability in different tissues of S. superba. Three software programs (geNorm, NormFinder, and BestKeeper) were used to evaluate the reference gene transcript stabilities, and comprehensive stability ranking was generated by the geometric mean method. Our results show that SsuACT was the most stable reference gene and that SsuACT + SsuRIB was the best reference gene combination for different tissues. Finally, the stable and less stable reference genes were verified using SsuSND1 expression in different tissues. To our knowledge, this is the first report to verify appropriate reference genes for normalizing gene expression in S. superba for different tissues, which will facilitate the future elucidation of gene regulations in this species and useful references for relative species.

Highlights

Plant gene expression analysis methods include Northern blot, in situ hybridization, RT-PCR, and real-time quantitative PCR (RT-qPCR)
RT-qPCR has been widely used in molecular biology research, and expression analysis is realized by real-time detection of fluorescence signal changes in the whole PCR reaction process due to its high sensitivity, accuracy, specificity, throughput capability, and cost-effectiveness [1,2,3,4,5]
Eighteen candidate reference genes were selected from the transcriptome of S. superba, and ColGAPDH was cited from Yang [22]

Summary

Introduction

Plant gene expression analysis methods include Northern blot, in situ hybridization, RT-PCR, and real-time quantitative PCR (RT-qPCR). RT-qPCR has been widely used in molecular biology research, and expression analysis is realized by real-time detection of fluorescence signal changes in the whole PCR reaction process due to its high sensitivity, accuracy, specificity, throughput capability, and cost-effectiveness [1,2,3,4,5]. The commonly used reference genes are mainly the genes that constitute the cytoskeleton or participate in the cells’ basic biochemical metabolic activities, including actin (ACT), β-tubulin (TUB), ribosomal RNA (18S rRNA, 26S rRNA), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ubiquitin (UBQ), and elongation factors 1 α (EF-1α) [8,9,10]. Studies have shown that the expression levels of these genes are specific and not stable across species, under different treatments. A reference gene suitable for all conditions does not exist [11,12,13]

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