The use of miRNAs as reference genes for miRNA expression normalization during Lilium somatic embryogenesis by real-time reverse transcription PCR analysis
The use of miRNAs as reference genes for miRNA expression normalization during Lilium somatic embryogenesis by real-time reverse transcription PCR analysis
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通过实时逆转录 PCR 分析，使用 miRNA 作为百合体细胞胚胎发生过程中 miRNA 表达标准化的参考基因
DOI：
10.1007/s11240-016-1160-9
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发表时间：
2017
期刊：
影响因子：
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通讯作者：
Sun HongMei
中科院分区：
文献类型：
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作者：
Zhang Jing;Gai MeiZhu;Xue BingYang;Jia Nana;Wang ChunXia;Wang JinXia;Sun HongMei
miRNAs的表达谱能够揭示体细胞胚胎发生的本质。qRT-PCR是最常用的动态miRNA检测技术之一，但需要最佳的参考基因以确保数据的可靠性。这是第一个在百合（Lilium pumilum DC.）中验证miRNA表达标准化的参考基因的报告。菲施。和兰州百合Lilium davidii var.单色）。本研究选择了17个miRNAs、2个snRNAs（U4、U6）、1个rRNA（5S rRNA）和3个蛋白质编码基因（FP、ACT、GAPDH）作为参考候选基因，并通过qRT-PCR验证了它们在2个百合SE发育过程中的表达稳定性。还使用了四种归一化算法，包括geNorm、BestKeeper、Normalization和Referral，来评估参考候选物的稳定性。对细叶百合（Lilium pumilum DC.）菲施。，lpu-miR 159 a是SE期间的最佳参考基因，其次是lpu-miR 408 b，而U6是最不稳定的参考候选基因。兰州百合Lilium davidii var.单色，FP比一半的miRNA候选者表现出更大的稳定性，但最好的参考基因是lda-miR 162，其次是lda-miR 159 a。进一步分析miR 156和miR 529的表达水平，以评估参考基因在两种百合中的有效性。 这是首次在百合属植物中使用合适的miRNAs作为参考基因，为进一步探索百合属植物中miRNAs的功能迈出了重要的一步，为其他植物的研究提供了有价值的参考。
Expression profiling of miRNAs has the ability to reveal the essence of somatic embryogenesis (SE). qRT-PCR is one of the most commonly used techniques for dynamic miRNA detection but requires optimal reference genes for data reliability. This is the first report on reference gene validation for miRNA expression normalization in Lilium (Lilium pumilum DC. Fisch. and Lilium davidii var. unicolor). In this study, seventeen miRNAs together with two snRNAs (U4, U6), one rRNA (5S rRNA) and three protein-coding genes (FP, ACT, GAPDH) were selected as reference candidates, and their expression stability was validated by qRT-PCR among eleven developing SE cultures in two lilies. Four normalization algorithms, including geNorm, BestKeeper, NormFinder and RefFinder, were also used to evaluate the stability of the reference candidates. For Lilium pumilum DC. Fisch., lpu-miR159a was the optimal reference gene during SE, followed by lpu-miR408b, while U6 was the least stable reference candidate. For Lilium davidii var. unicolor, FP presented greater stability than did half of the miRNA candidates, but the best reference gene was lda-miR162, followed by lda-miR159a. Further analysis of the expression level of miR156 and miR529 was used to evaluate the validity of the reference genes in both lilies. This is a pioneer study using suitable miRNAs as reference genes in Lilium and constitutes a small but essential step for the further exploration of miRNA function in Lilium, thus offering valuable references for other plants.