A simplified and optimal RT-qPCR strategy for extracellular miRNA profiling

Introduction: Extracellular miRNAs in the blood plasma/serum of patients as novel promising diagnostic markers for various diseases has aroused huge attention. Among various major platforms for extracellular miRNA detection, extensively used RT-qPCR is considered practical and cost-efficient in experimental investigation. However, there are also many technical challenges, hence the development of robust methodology for extracellular miRNA detection is in good need. Methods: Focused on the controversial procedures present in recent studies, we intend to solving these technical problems and seeking for optimal procedures for extracellular miRNA profiling using the RT-qPCR method. Results: We addressed our concerns during the whole detection such as various key points during blood handling, choice making during RNA isolation and miRNA RT process, practical management in global miRNAs profiling using PCR as well as data normalization and processing. Meanwhile, we described and evaluated our profiling method in detail, intended to optimize the procedures that might lead to variable results from independent experiments, and further recommend this standardized optimal procedure. Conclusion: Our work might provide a potent technical optimization for extracellular miRNA detection.