College of Horticulture, Fujian Agriculture and Forestry University, Fuijan 350002, China.
Department of Environmental Horticulture and Mid-Florida Research and Education Center, Institute of Food and Agricultural Sciences, University of Florida, Apopka, FL 32703, USA.
Genes (Basel). 2019 Dec 23;11(1):17. doi: 10.3390/genes11010017.
MicroRNAs (miRNAs) are short noncoding RNA molecules that regulate gene expression at the posttranscriptional level. Reverse transcription-quantitative PCR (RT-qPCR) is one of the most common methods used for quantification of miRNA expression, and the levels of expression are normalized by comparing with reference genes. Thus, the selection of reference genes is critically important for accurate quantification. The present study was intended to identify appropriate miRNA reference genes for normalizing the level of miRNA expression in L. Osbeck and Blanco infected by subsp. , which caused citrus canker disease. Five algorithms (Delta Ct, geNorm, NormFinder, BestKeeper and RefFinder) were used for screening reference genes, and two quantification approaches, poly(A) extension RT-qPCR and stem-loop RT-qPCR, were used to determine the most appropriate method for detecting expression patterns of miRNA. An overall comprehensive ranking output derived from the multi-algorithms showed that poly(A)-tailed miR162-3p/miR472 were the best reference gene combination for miRNA RT-qPCR normalization in citrus canker research. Candidate reference gene expression profiles determined by poly(A) RT-qPCR were more consistent in the two citrus species. To the best of our knowledge, this is the first systematic comparison of two miRNA quantification methods for evaluating reference genes. These results highlight the importance of rigorously assessing candidate reference genes and clarify some contradictory results in miRNA research on citrus.
MicroRNAs (miRNAs) 是短的非编码 RNA 分子，可在转录后水平调控基因表达。逆转录定量聚合酶链式反应 (RT-qPCR) 是最常用的 miRNA 表达定量方法之一，通过与参照基因比较来对表达水平进行标准化。因此，参照基因的选择对于准确的定量至关重要。本研究旨在鉴定合适的 miRNA 参照基因，以标准化 L. Osbeck 和 Blanco 被引起柑橘溃疡病的 subsp. 感染后的 miRNA 表达水平。使用了五种算法（Delta Ct、geNorm、NormFinder、BestKeeper 和 RefFinder）来筛选参照基因，并使用 Poly(A) 延伸 RT-qPCR 和茎环 RT-qPCR 两种定量方法来确定检测 miRNA 表达模式的最适方法。多算法综合得出的全面排名结果表明，Poly(A) 尾巴的 miR162-3p/miR472 是柑橘溃疡病研究中 miRNA RT-qPCR 标准化的最佳参照基因组合。通过 Poly(A) RT-qPCR 确定的候选参照基因表达谱在两种柑橘物种中更为一致。据我们所知，这是首次对两种 miRNA 定量方法进行系统比较，以评估参照基因。这些结果强调了严格评估候选参照基因的重要性，并澄清了柑橘上 miRNA 研究中的一些矛盾结果。
