MicroRNAs In Single Cells Were Detected By Microtiter Digital PCR Based On Ligation Reaction

MicroRNAs are a class of endogenous and nonprotein coding short RNA generally containing about 19-25 nucleotides, which play crucial roles in regulation of biological progress such as cell proliferation, migration and apoptosis. The aberrant expression of some microRNAs is associated with various diseases, so microRNAs have been an emerging important biomarkers for early clinical diagnostics. However, small size, sequence homology and relatively low expression levels in cells make the microRNA detection difficult. Recently, microRNA detection is attracting extensive attentions owing to its great significance for human disease diagnosis and treatment as well as for fundamental biological research. However, the highly sensitive, specific, especially accurate and quantitative microRNA detection still remains great challenges. On the other hand, the cell-to-cell variations of gene expression in mammalian cells, the important information connecting microRNA expression and cell function may be masked by ensemble measurements of microRNAs in cell populations. Therefore, the development of simple, accurate and sensitive method for quantitative detection of microRNAs, especially in a single cell, is still a very high requirement.In this paper, taking the advantages of the high specificity and simpleness of the ligation-based PCR, and the precise quantification and high sensitivity of ddPCR, we study a novel assay for precise quantification of microRNAs. The probe A and probe B are simply designed to be complementary to the half-sequence of target microRNA with two ribonucleotides and phosphate group at the 3'and 5'end. In the presence of the target microRNAs, the probe A and probe B hybridize with the target and then can be ligated with each other under the catalysis of T4 RNA ligase 2. With the ddPCR amplification of the ligation products, microRNA can be quantitatively detected in the extremely low abundance with high sensitivity (as low as 20 aM microRNA can be detected) and specificity, and linear range spans over four orders of magnitude, which also promises the precise quantitation of copy numbers of microRNA in a single cell. The assay may act as a powerful tool to allow for deep insight into how microRNAs influence the central signalling pathways and the cell functions at a single cell level, which are of great significance for microRNA-related clinical studies.