Validation of Novel Reference Genes in Different Rice Plant Tissues through Mining RNA-Seq Datasets

Abstract

Reverse transcription quantitative real-time PCR (RT-qPCR) is arguably the most prevalent and accurate quantitative gene expression analysis. However, selection of reliable reference genes for RT-qPCR in rice (Oryza sativa) is still limited, especially for a specific tissue type or growth condition. In this study, we took the advantage of our RNA-seq datasets encompassing data from five rice varieties with diverse treatment conditions, identified 12 novel candidate reference genes, and conducted rigorous evaluations of their suitability across typical rice tissues. Comprehensive analysis of the leaves, shoots, and roots of two rice seedlings subjected to salt (30 mmol/L NaCl) and drought (air-dry) stresses have revealed that OsMED7, OsACT1, and OsOS-9 were the robust reference genes for leaf samples, while OsACT1, OsZOS3-23, and OsGDCP were recommended for shoots and OsMED7, OsOS-9, and OsGDCP were the most reliable reference genes for roots. Comparison results produced by different sets of reference genes revealed that all these newly recommended reference genes displayed less variation than previous commonly used references genes under the experiment conditions. Thus, selecting appropriate reference genes from RNA-seq datasets leads to identification of reference genes suitable for respective rice tissues under drought and salt stress. The findings offer valuable insights for refining the screening of candidate reference genes under diverse conditions through the RNA-seq database. This refinement serves to improve the accuracy of gene expression in rice under similar conditions.

Evaluation of Suitable Reference Genes for Quantitative Real-Time PCR in Various Tissues of Apocynum venetum

“…The two most stable reference genes in leaves, stems, and roots were CYP and TUA, PGK and PPP2R3, and TUA and EF-1α, respectively. Similar results of varied expressions in different tissues have been reported in some plant species [4,9,28]. From the above results, we also found that the expression levels of reference genes were not always stable across different tissues in A. venetum.…”

“…Quantification of specific mRNA is a fundamental requirement for analyzing gene expression. At present, Northern blotting, microarray analysis, quantitative real-time PCR (qRT-PCR), and RNA sequencing are employed to quantify specific mRNA levels in diverse tissues of an organism [2][3][4]. Among them, qRT-PCR emerges as a highly dependable technique for analyzing gene expression, primarily owing to its sensitivity, reproducibility, specificity, and capacity for high yield [5,6].…”