Instituto Politécnico Nacional, Centro de Investigación en Biotecnología Aplicada (CIBA-IPN), Ex-Hacienda San Juan Molino Carretera Estatal Tecuexcomac-Tepetitla, Tlaxcala, México.
Plant Biol (Stuttg). 2018 Jul;20(4):713-721. doi: 10.1111/plb.12725. Epub 2018 Apr 17.
Studies of gene expression are very important for the identification of genes that participate in different biological processes. Currently, reverse transcription quantitative real-time PCR (RT-qPCR) is a high-throughput, sensitive and widely used method for gene expression analysis. Nevertheless, RT-qPCR requires precise normalisation of data to avoid the misinterpretation of experimental data. In this sense, the selection of reference genes is critical for gene expression analysis. At this time, several studies focus on the selection of reference genes in several species. However, the identification and validation of reference genes for the normalisation of RT-qPCR have not been described in amaranth. A set of seven housekeeping genes were analysed using RT-qPCR, to determine the most stable reference genes in amaranth for normalisation of gene expression analysis. Transcript stability and gene expression level of candidate reference genes were analysed in different tissues, at different developmental stages and under different types of stress. The data were compared using the geNorm, NormFinder and Bestkeeper statistical methods. The reference genes optimum for normalisation of data varied with respect to treatment. The results indicate that AhyMDH, AhyGAPDH, AhyEF-1α and AhyACT would be optimum for accurate normalisation of experimental data, when all treatment are analysed in the same experiment. This study presents the most stable reference genes for normalisation of gene expression analysis in amaranth, which will contribute significantly to future gene studies of this species.
基因表达研究对于鉴定参与不同生物过程的基因非常重要。目前，逆转录定量实时 PCR（RT-qPCR）是一种高通量、敏感且广泛应用的基因表达分析方法。然而，RT-qPCR 需要对数据进行精确的归一化，以避免对实验数据的误解。在这种情况下，选择参考基因对于基因表达分析至关重要。目前，已有多项研究致力于在多个物种中选择参考基因。然而，在苋菜中，尚未对 RT-qPCR 归一化的参考基因进行鉴定和验证。本研究使用 RT-qPCR 分析了 7 个管家基因，以确定苋菜中最稳定的参考基因，用于归一化基因表达分析。候选参考基因的转录稳定性和基因表达水平在不同组织、不同发育阶段和不同类型的胁迫下进行了分析。使用 geNorm、NormFinder 和 Bestkeeper 统计方法比较了数据。最佳参考基因因处理而异。结果表明，当在同一实验中分析所有处理时，AhyMDH、AhyGAPDH、AhyEF-1α 和 AhyACT 是准确归一化实验数据的最佳选择。本研究为苋菜基因表达分析提供了最稳定的参考基因，这将对该物种的未来基因研究有重大贡献。
