The discovery of microRNAs (miRNAs) added an extra level of intricacy to the already complex system regulating gene expression. These single-stranded RNA molecules, 18-25 nucleotides in length, negatively regulate gene expression through translational inhibition or mRNA cleavage. The discovery that aberrant expression of specific miRNAs contributes to human disease has fueled much interest in profiling the expression of these molecules. Real-time quantitative PCR (RQ-PCR) is a sensitive and reproducible gene expression quantitation technique which is now being used to profile miRNA expression in cells and tissues. To correct for systematic variables such as amount of starting template, RNA quality and enzymatic efficiencies, RQ-PCR data is commonly normalised to an endogenous control (EC) gene, which ideally, is stably-expressed across the test sample set. A universal endogenous control suitable for every tissue type, treatment and disease stage has not been identified and is unlikely to exist, so, to avoid introducing further error in the quantification of expression data it is necessary that candidate ECs be validated in the samples of interest. While ECs have been validated for quantification of mRNA expression in various experimental settings, to date there is no report of the validation of miRNA ECs for expression profiling in breast tissue. In this study, the expression of five miRNA genes (let-7a, miR-10b, miR-16, miR-21 and miR-26b) and three small nucleolar RNA genes (RNU19, RNU48 and Z30) was examined across malignant, benign and normal breast tissues to determine the most appropriate normalisation strategy. This is the first study to identify reliable ECs for analysis of miRNA by RQ-PCR in human breast tissue.

中文翻译：

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鉴定合适的内源性控制基因，用于人乳腺癌中的 microRNA 基因表达分析。

microRNA (miRNA) 的发现为已经复杂的基因表达调控系统增添了额外的复杂性。这些长度为 18-25 个核苷酸的单链 RNA 分子通过翻译抑制或 mRNA 切割负向调节基因表达。特定 miRNA 的异常表达导致人类疾病的发现激起了人们对分析这些分子表达的兴趣。实时定量 PCR (RQ-PCR) 是一种灵敏且可重复的基因表达定量技术，现在用于分析细胞和组织中的 miRNA 表达。为了校正系统变量，如起始模板量、RNA 质量和酶促效率，RQ-PCR 数据通常被标准化为内源性控制 (EC) 基因，理想情况下，在测试样本集上稳定表达。适用于每种组织类型、治疗和疾病阶段的通用内源性对照尚未确定并且不太可能存在，因此，为了避免在表达数据的量化中引入进一步的错误，有必要在感兴趣的样本中验证候选 EC . 虽然 ECs 已经在各种实验环境中被验证用于 mRNA 表达的量化，但迄今为止还没有关于 miRNA ECs 用于乳腺组织中表达谱的验证的报告。在本研究中，检测了恶性、良性、恶性和良性的 5 个 miRNA 基因（let-7a、miR-10b、miR-16、miR-21 和 miR-26b）和三个小核仁 RNA 基因（RNU19、RNU48 和 Z30）的表达。和正常乳房组织，以确定最合适的标准化策略。