Simple And Sensitive Detection Of MicroRNA With Ligase Chain Reaction

MicroRNA (miRNA) play an important regulatory role in biological systems. Studies of the biological functions of miRNA are have great potential for early clinical diagnosis and diacovery of new drugs. Developing simple, rapid, highly sensitive and specific methods for miRNAs detection are very important for microRNA studies. In this dissertation, by using miRNAs as a template, DNA probes immobilized on different gold nanoparticles were successfully ligated, which resulted in the cross-linking aggregation of gold nanoparticles. Therefore miRNAs can be simply detected by visualization of the color changes. This method was simple and easy to operate, and does not need any method of amplification and expensive equipment. The results of this method can be distinguished with naked eyes, which facilitated its application. The limitation of the proposed method is that it is difficult to carry out the ligase chain reaction (LCR), so the sensitivity is not satisfactory. On this basis, we developed a simple and sensitive method, in which let-7a miRNA was used as the template and T4 RNA ligase 2 was used to selectively ligate DNA probes. Thus the LCR was initiated and the products were separated and detected by agarose gel electrophoresis. The detection limit (3σ, n = 7) of Let-7a was estimated to be 0.7 pmol/L. There is a good correlative relationship between the pixel intensity of LCR products and the concentration of let-7a in the range of 2.0 pMB2.0 nM. Due to the high specificity of the LCR reaction, the LCR miRNA assay can clearly discriminate one base difference between miRNA sequences. The LCR assay has been applied to the quantitation of let-7a content in Human lung total RNA sample. This method can be universally applicable for various miRNAs analysis upon designing suitable probes. Therefore, the LCR method has a good prospect in detection of miRNAs.