BACKGROUND MicroRNAs (miRNAs) are small endogenous non-coding interfering RNA molecules regarded as major regulators in eukaryotic gene expression. Different methods are employed for miRNA expression profiling. For a better understanding of their role in essential biological processes, convenient methods for differential miRNA expression analysis are required. RESULTS Here, we present the miR-Q assay as a highly sensitive quantitative reverse transcription PCR (qRT-PCR) for expression analysis of small RNAs such as miRNA molecules. It shows a high dynamic range of 6 to 8 orders of magnitude comprising a sensitivity of up to 0.2 fM miRNA, which corresponds to single copies per cell. There is nearly no cross reaction among closely-related miRNA family members, which points to the high specificity of the assays. Using this approach, we quantified the expression of let-7b in different human cell lines as well as miR-145 and miR-21 expression in porcine intestinal samples. CONCLUSION miR-Q is a cost-effective and highly specific approach, which neither requires the use of fluorochromic probes, nor Locked Nucleic Acid (LNA)-modified oligonucleotides. Moreover, it provides a remarkable increase in specificity and simplified detection of small RNAs.

中文翻译：

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miR-Q：一种新颖的定量 RT-PCR 方法，用于对复杂样品中的小 RNA 分子（如 miRNA）进行表达分析。

背景微RNA (miRNA) 是小的内源性非编码干扰RNA 分子，被认为是真核基因表达的主要调节剂。不同的方法用于 miRNA 表达谱。为了更好地了解它们在基本生物过程中的作用，需要使用方便的方法进行差异 miRNA 表达分析。结果 在这里，我们将 miR-Q 检测作为一种高度灵敏的定量逆转录 PCR (qRT-PCR) 来表达，用于小 RNA 的表达分析，例如 miRNA 分子。它显示了 6 到 8 个数量级的高动态范围，包括高达 0.2 fM miRNA 的灵敏度，这对应于每个细胞的单个拷贝。密切相关的 miRNA 家族成员之间几乎没有交叉反应，这表明检测具有高度的特异性。使用这种方法，我们量化了 let-7b 在不同人类细胞系中的表达以及 miR-145 和 miR-21 在猪肠道样本中的表达。结论 miR-Q 是一种经济高效且高度特异性的方法，既不需要使用荧光变色探针，也不需要使用锁核酸 (LNA) 修饰的寡核苷酸。此外，它显着提高了小 RNA 的特异性并简化了检测。