Laboratory of Experimental Biochemistry & Molecular Biology, IRCCS Istituto Ortopedico Galeazzi, Milano, Italy.
Laboratory of Experimental Biochemistry & Molecular Biology, IRCCS Istituto Ortopedico Galeazzi, Milano, Italy; Vita-Salute San Raffaele University, Milano, Italy.
Adv Clin Chem. 2018;87:113-139. doi: 10.1016/bs.acc.2018.07.003. Epub 2018 Aug 23.
Circulating molecules that are released into the circulation in response to specific stimuli are considered potential biomarkers for physiological or pathological processes. Their effective usefulness as biomarkers resides in their stability and high availability in all the biological fluids, combined with the limited invasiveness of intervention. Among the circulating molecules, miRNAs represent a novel class of biomarkers as they possess all the required characteristics such as sensitivity, predictivity, specificity, robustness, translatability, and noninvasiveness. miRNAs are small non-coding RNAs, that act as inhibitors of protein translation, and intervene in the complex network of the post-transcriptional mechanisms finely regulating gene expression. The emerging role of miRNAs as potential biomarkers for clinical applications (e.g., cancer and cardiovascular diseases diagnosis and prediction, musculoskeletal disease diagnosis and bone fracture risk prediction), however, requires the standardization of miRNA processing, from sample collection and sample storage, to RNA isolation, RNA reverse-transcription, and data analyses. Normalization is one of the most controversial issues related to quantitative Real-Time PCR data analysis since no universally accepted normalization strategies and reference genes exist, even more importantly, for circulating miRNA quantification. As it is widely demonstrated that the choice of different normalization strategies influences the results of gene expression analysis, it is important to select the most appropriate normalizers for each experimental set. This review discloses on the different strategies adopted in RT-qPCR miRNA normalization and the concerning issues to highlight on the need of a universally accepted methodology to make comparable the results produced by different studies.
循环分子是在特定刺激下释放到循环系统中的分子，被认为是生理或病理过程的潜在生物标志物。它们作为生物标志物的有效性取决于它们在所有生物流体中的稳定性和高可用性，以及干预的有限侵入性。在循环分子中，miRNAs 是一类新型的生物标志物，因为它们具有所有必需的特征，如敏感性、预测性、特异性、稳健性、可翻译性和非侵入性。miRNAs 是小的非编码 RNA，作为蛋白质翻译的抑制剂，参与转录后机制的复杂网络，精细调节基因表达。miRNAs 作为临床应用（如癌症和心血管疾病的诊断和预测、肌肉骨骼疾病的诊断和骨折风险预测）的潜在生物标志物的新兴作用，然而，需要标准化 miRNA 的处理，从样本收集和样本储存到 RNA 分离、RNA 逆转录和数据分析。标准化是与定量实时 PCR 数据分析相关的最具争议的问题之一，因为即使对于循环 miRNA 的定量，也没有普遍接受的标准化策略和参考基因。由于广泛证明不同的标准化策略会影响基因表达分析的结果，因此选择最适合每个实验集的归一化器非常重要。这篇综述揭示了 RT-qPCR miRNA 归一化中采用的不同策略以及需要普遍接受的方法学来比较不同研究产生的结果的关注问题。
