Department of Physiology and Cell Biology, University of Nevada, Reno School of Medicine, Reno, Nevada, USA.
Department of Biology, University of Nevada, Reno School of Medicine, Reno, Nevada, USA.
Biol Reprod. 2017 Sep 1;97(3):490-496. doi: 10.1093/biolre/iox102.
Quantitative analyses of small RNAs at the single-cell level have been challenging because of limited sensitivity and specificity of conventional real-time quantitative PCR methods. A digital quantitative PCR (dqPCR) method for miRNA quantification has been developed, but it requires the use of proprietary stem-loop primers and only applies to miRNA quantification. Here, we report a microfluidics-based dqPCR (mdqPCR) method, which takes advantage of the Fluidigm BioMark HD system for both template partition and the subsequent high-throughput dqPCR. Our mdqPCR method demonstrated excellent sensitivity and reproducibility suitable for quantitative analyses of not only miRNAs but also all other small RNA species at the single-cell level. Using this method, we discovered that each sperm has a unique miRNA profile.
单细胞水平的小 RNA 定量分析一直具有挑战性，因为传统的实时定量 PCR 方法的灵敏度和特异性有限。已经开发了一种用于 miRNA 定量的数字定量 PCR（dqPCR）方法，但它需要使用专有的茎环引物，并且仅适用于 miRNA 定量。在这里，我们报告了一种基于微流控的 dqPCR（mdqPCR）方法，该方法利用 Fluidigm BioMark HD 系统进行模板分区和随后的高通量 dqPCR。我们的 mdqPCR 方法表现出优异的灵敏度和重现性，不仅适用于 miRNA 的定量分析，还适用于单细胞水平上所有其他小 RNA 种类的定量分析。使用这种方法，我们发现每个精子都有独特的 miRNA 图谱。
