Beijing Advanced Innovation Center for Tree Breeding By Molecular Design, College of Biological Sciences and Biotechnology, National Engineering Laboratory for Tree Breeding, Beijing Forestry University, Beijing, 100083, China.
Institute of Pomology, Jiangsu Key Laboratory for Horticultural Crop Genetic Improvement, Jiangsu Academy of Agricultural Sciences, Nanjing, 200014, China.
Mol Biol Rep. 2021 Apr;48(4):3357-3366. doi: 10.1007/s11033-021-06369-y. Epub 2021 May 4.
Quantitative real time polymerase chain reaction (qRT-PCR) is a common method to analyze gene expression. Due to differences in RNA quantity, quality, and reverse transcription efficiency between qRT-PCR samples, reference genes are used as internal standards to normalize gene expression. However, few universal genes, especially miRNAs, have been identified as reference so far. Therefore, it is essential to identify reference genes that can be used across various experimental conditions, stress treatments, or tissues. In this study, 14 microRNAs (miRNAs) and 5.8S rRNA were assessed for expression stability in poplar trees infected with canker pathogen. Using geNorm, NormFinder and Bestkeeper reference gene analysis programs, we found that miR156g and miR156a exhibited stable expression throughout the infection process. miR156g, miR156a and 5.8S rRNA were then tested as internal standards to measure the expression of miR1447 and miR171c, and the results were compared to small RNA sequencing (RNA-seq) data. We found that when miR156a and 5.8S rRNA were used as the reference gene, the expression of miR1447 and miR171c were consistent with the small RNA-seq expression profiles. Therefore, miR156a was the most stable miRNAs examined in this study, and could be used as a reference gene in poplar under canker pathogen stress, which should enable comprehensive comparisons of miRNAs expression and avoid the bias caused by different length between detected miRNAs and traditional reference genes. The present study has expanded the miRNA reference genes available for gene expression studies in trees under biotic stress.
实时荧光定量聚合酶链反应（qRT-PCR）是分析基因表达的常用方法。由于 qRT-PCR 样品之间的 RNA 数量、质量和逆转录效率存在差异，因此使用参照基因作为内参来标准化基因表达。然而，迄今为止，很少有通用基因（特别是 miRNA）被鉴定为参照。因此，确定可在各种实验条件、应激处理或组织中使用的参照基因至关重要。在这项研究中，评估了 14 种 microRNAs（miRNAs）和 5.8S rRNA 在感染溃疡病原菌的杨树中的表达稳定性。使用 geNorm、NormFinder 和 BestKeeper 参照基因分析程序，我们发现 miR156g 和 miR156a 在整个感染过程中表达稳定。然后将 miR156g、miR156a 和 5.8S rRNA 用作内参来测量 miR1447 和 miR171c 的表达，并将结果与小 RNA 测序（RNA-seq）数据进行比较。我们发现，当使用 miR156a 和 5.8S rRNA 作为参照基因时，miR1447 和 miR171c 的表达与小 RNA-seq 表达谱一致。因此，miR156a 是本研究中最稳定的 miRNA，可以作为溃疡病原菌胁迫下杨树的参照基因，这将能够全面比较 miRNAs 的表达，并避免由于检测的 miRNAs 与传统参照基因之间的长度不同而导致的偏差。本研究扩展了可用于生物胁迫下树木基因表达研究的 miRNA 参照基因。
