Shanghai Innostar Bio-tech Co. Ltd., China State Institute of Pharmaceutical Industry, Shanghai, People's Republic of China.
Vet Med Sci. 2022 Sep;8(5):2076-2085. doi: 10.1002/vms3.879. Epub 2022 Jul 27.
In quantitative reverse transcription-polymerase chain reaction (RT-qPCR) studies, the selection and validation of reference genes are crucial for the accurate analysis of MicroRNAs (miRNAs) expression. In this work, the optimal reference genes for RT-qPCR normalisation in plasma samples of rat middle cerebral artery occlusion (MCAO) models were identified.
Six rat MCAO models were established. Blood samples were collected before modelling and approximately 16-24 h after modelling. Two commonly used reference genes (U6 and 5S) and three miRNAs (miR-24, miR-122 and miR-9a) were selected as candidate reference genes, and the expression of these genes was detected with RT-qPCR. The acquired data were analysed using geNorm, Normfinder, BestKeeper, RefFinder and comparative delta threshold cycle statistical models.
The analysed results consistently showed that miR-24 was the most stably expressed reference gene. The 'optimal combination' calculated by geNorm was miR-24, U6 and5S. The expression level of the target gene miR124 was similar when the most stable reference gene miR-24 or the 'optimal combination' was used as a reference gene. However, compared with miR24 or the 'optimal combination', the less stable reference genes influenced the fold change and the data accuracy with a large standard deviation.
These results confirmed the importance of selecting suitable reference genes for normalisation to obtain reliable results in RT-qPCR studies and demonstrated that the identified reference gene miR-24 or the 'optimal combination' could be used as an internal control for gene expression analysis in the rat MCAO model.
在定量逆转录聚合酶链反应（RT-qPCR）研究中，选择和验证参考基因对于准确分析 microRNAs（miRNAs）表达至关重要。本研究旨在确定大鼠大脑中动脉闭塞（MCAO）模型血浆样本中 RT-qPCR 归一化的最佳参考基因。
建立了 6 个大鼠 MCAO 模型。在建模前和建模后约 16-24 小时采集血液样本。选择了两个常用的参考基因（U6 和 5S）和三个 miRNAs（miR-24、miR-122 和 miR-9a）作为候选参考基因，并通过 RT-qPCR 检测这些基因的表达。使用 geNorm、Normfinder、BestKeeper、RefFinder 和比较 delta 阈值循环统计模型分析获得的数据。
分析结果一致表明，miR-24 是最稳定表达的参考基因。geNorm 计算的“最佳组合”是 miR-24、U6 和 5S。当使用最稳定的参考基因 miR-24 或“最佳组合”作为参考基因时，靶基因 miR124 的表达水平相似。然而，与 miR24 或“最佳组合”相比，稳定性较低的参考基因会影响 fold change 和数据准确性，导致较大的标准偏差。
这些结果证实了在 RT-qPCR 研究中选择合适的参考基因进行归一化以获得可靠结果的重要性，并表明鉴定的参考基因 miR-24 或“最佳组合”可用于大鼠 MCAO 模型中基因表达分析的内参。
