Background

microRNAs (miRNAs) are small non-coding RNAs regulating gene expression. They have attracted significant interest as biomarkers for early diagnosis, prediction and monitoring of treatment response in many diseases. As individual miRNAs often lack the required sensitivity and specificity, miRNA signatures are developed for clinical applications. Digital PCR (dPCR) is a sensitive fluorescent-based quantification method, that can be used to detect the expression of miRNAs in patient samples. Our study presents the first proof-of-concept of a multiplexed dPCR assay for the simultaneous analysis and quantification of multiple miRNAs.

Results

After reverse transcription (RT) using a pool of miRNA-specific stem-loop primers, dPCR was performed with a universal reverse primer and miRNA-specific forward primers along with fluorescently-labelled hydrolysis probes. Multiple experimental parameters were evaluated and strategies for modulating the observed signals were devised. The optimised assay was applied to the analysis of miRNAs from cell lines and biological samples. Although absolute quantification was lost, due to the reverse transcription step, quantification was linear for the dilution series and results were highly reproducible for independent dPCR and RT reactions. Our results confirmed the high sensitivity of dPCR for patient samples.

Conclusions

We demonstrate the feasibility and reliability of multiplexed detection and quantification of miRNAs by dPCR that can be applied in a clinical setting to evaluate miRNA signatures.

中文翻译：

---

用于同时定量 miRNA panel 的多重数字 PCR

 背景

microRNA （miRNA） 是调节基因表达的小非编码 RNA。它们作为许多疾病的早期诊断、预测和监测治疗反应的生物标志物引起了人们的极大兴趣。由于单个 miRNA 通常缺乏所需的灵敏度和特异性，因此为临床应用开发了 miRNA 特征。数字 PCR （dPCR） 是一种灵敏的基于荧光的定量方法，可用于检测患者样本中 miRNA 的表达。我们的研究提出了第一个用于同时分析和定量多个 miRNA 的多重 dPCR 检测的概念验证。

 结果

使用一组 miRNA 特异性茎环引物进行逆转录 （RT） 后，使用通用反向引物和 miRNA 特异性正向引物以及荧光标记的水解探针进行 dPCR。评估了多个实验参数并设计了调制观察到的信号的策略。优化的分析方法应用于细胞系和生物样品中的 miRNA 分析。尽管由于逆转录步骤而损失了绝对定量，但稀释系列的定量结果是线性的，并且独立 dPCR 和 RT 反应的结果具有高度重现性。我们的结果证实了 dPCR 对患者样本的高灵敏度。

 结论

我们证明了通过 dPCR 对 miRNA 进行多重检测和定量的可行性和可靠性，这些检测和定量可用于临床环境以评估 miRNA 特征。