A Highly Multiplexed Real-Time PCR for Quantitative Profiling of RNAs from Limited Clinical Specimens

摘要

We have developed quasi-liquid particles for real-time PCR and enabled multiplex quantification of miRNAs in 10 μL of sample. Each particle contained specific primers for a miRNA and marked with a specific pattern. Since extracelluar vesicles are very tricky to collect and analyze in traditional ways, multiplex detection of tens of genetic targets is critical to have a holistic view of the sample. The particle-based qPCR was improved in stability and reliability. Ten particles of individual primers were mixed in master mix and simply analyzed according to the general work flow of real time PCR. Single strike of the assay provided quantitative information of the ten target miRNAs. The reliability of the multiplex assay was validated through comparison with single-plex assays. The multiplicity of the assay is 20 target at a time in the present platform.