Expression Stabilities of Candidate Reference Genes for RT-qPCR in Chinese Jujube (Ziziphus jujuba Mill.) under a Variety of Conditions.
Expression Stabilities of Candidate Reference Genes for RT-qPCR in Chinese Jujube (Ziziphus jujuba Mill.) under a Variety of Conditions.
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DOI：
10.1371/journal.pone.0154212
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发表时间：
2016
期刊：
影响因子：
3.7
通讯作者：
Liu M
中科院分区：
文献类型：
--
作者：
Bu J;Zhao J;Liu M
逆转录定量实时聚合酶链反应 (RT-qPCR) 是评估基因表达模式的强大方法。枣全基因组测序已经完成，基因功能分析是后续研究的重要组成部分，需要选择合适的内参基因。事实上，RT-qPCR 中合适的参考基因选择对于靶基因表达的准确标准化至关重要。本研究利用软件包geNorm和NormFinder检测了9个候选内参基因在多种条件下的表达稳定性。肌动蛋白解聚因子 1 (ACT1)、组蛋白-H3 (His3) 和多腺苷酸结合蛋白相互作用蛋白 (PAIP) 被确定为果实发育五个阶段中最稳定表达的基因，ACT1、SiR-Fd、BTF3 和微管蛋白 α 链 (TUA) 在不同组织/器官中表达最稳定。而 ACT1、碱性转录因子 3 (BTF3)、3-磷酸甘油醛脱氢酶 (GADPH) 和 PAIP 在黑暗条件下最稳定。 ACT1、PAIP、BTF3 和延伸因子 1-γ (EF1γ) 是植原体感染下表达最稳定的基因。在这些基因中，SiR-Fd 和 PAIP 首次被报道为稳定的参考基因。当使用这些最稳定的参考基因进行归一化时，发现四个目标基因的表达模式与生理数据一致，表明我们研究中选择的参考基因适合用于此类分析。本研究为进一步的枣RT-qPCR研究提供了合适的内参基因和相应的引物，并强调了在可变实验条件下验证内参基因对于基因表达分析的重要性。
Reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) is a powerful method for evaluating patterns of gene expression. Jujube whole-genome sequencing has been completed, and analysis of gene function, an important part of any follow-up study, requires the appropriate selection of reference genes. Indeed, suitable reference gene selection for RT-qPCR is critical for accurate normalization of target gene expression. In this study, the software packages geNorm and NormFinder were employed to examine the expression stabilities of nine candidate reference genes under a variety of conditions. Actin-depolymerizing factor 1 (ACT1), Histone-H3 (His3), and Polyadenylate-binding protein-interacting protein (PAIP) were determined to be the most stably expressed genes during five stages of fruit development and ACT1, SiR-Fd, BTF3, and Tubulin alpha chain (TUA) across different tissues/organs. Whereas ACT1, Basic Transcription factor 3 (BTF3), Glyceraldehyde-3-phosphate dehydrogenase (GADPH), and PAIP were the most stable under dark conditions. ACT1, PAIP, BTF3, and Elongation factor 1- gamma (EF1γ) were the most stably expressed genes under phytoplasma infection. Among these genes, SiR-Fd and PAIP are here first reported as stable reference genes. When normalized using these most stable reference genes, the expression patterns of four target genes were found to be in accordance with physiological data, indicating that the reference genes selected in our study are suitable for use in such analyses. This study provides appropriate reference genes and corresponding primers for further RT-qPCR studies in Chinese jujube and emphasizes the importance of validating reference genes for gene expression analysis under variable experimental conditions.