Department of Biotechnology, College of Agriculture, Isfahan University of Technology, Isfahan, Iran.
Sci Rep. 2024 Sep 27;14(1):22113. doi: 10.1038/s41598-024-73444-2.
Accurate measurement of gene expression levels is vital for advancing plant biology research. This study explores the identification and validation of stable reference genes (RGs) for gene expression analysis in Spinacia oleracea. Leveraging transcriptome data from various developmental stages, we employed rigorous statistical analyses to identify potential RGs. A total of 1196 candidate genes were initially screened based on expression variability, with subsequent refinement using criteria such as low variance and stability. Among 12 commonly used candidate RGs, EF1α and H3 emerged as the most stable across diverse experimental conditions, while GRP and PPR exhibited lower stability. These findings were further validated through qRT-PCR assays and comprehensive statistical analyses, including geNorm, NormFinder, BestKeeper, and RefFinder. Our study underscores the importance of systematic RG selection to ensure accurate normalization in gene expression studies, particularly in the context of S. oleracea developmental stages and physiological processes like flowering. These validated RGs provide a robust foundation for future gene expression analysis in S. oleracea and contribute to the advancement of molecular research in plant biology.
准确测量基因表达水平对于推进植物生物学研究至关重要。本研究旨在探索和验证菠菜中用于基因表达分析的稳定参考基因（RGs）。本研究利用来自不同发育阶段的转录组数据，通过严格的统计分析来鉴定潜在的 RGs。最初根据表达变异性筛选了 1196 个候选基因，然后根据低方差和稳定性等标准进行了进一步的筛选。在 12 个常用的候选 RG 中，EF1α 和 H3 在各种实验条件下表现出最高的稳定性，而 GRP 和 PPR 则表现出较低的稳定性。通过 qRT-PCR 分析和全面的统计分析（包括 geNorm、NormFinder、BestKeeper 和 RefFinder）进一步验证了这些发现。本研究强调了系统选择 RG 的重要性，以确保在基因表达研究中进行准确的归一化，特别是在菠菜发育阶段和开花等生理过程的背景下。这些经过验证的 RG 为菠菜的未来基因表达分析提供了可靠的基础，并为植物生物学的分子研究进展做出了贡献。
