Sensitive Detection Of Micrornas With Loop-mediated Isothermal Amplification (LAMP)

MicroRNAs (miRNAs), a class of endogenous, nonprotein-coding small RNAs, play important roles in gene expression regulation by the formation of an RNA-induced silencing complex (RISC) with target mRNAs, which have critical functions in a wide range of physiologic and pathologic processes. Recent works have also revealed that miRNA expression can informatively reflect the developmental lineage and differentiation state of the tumours. miRNAs are attractive candidates of biomarkers for early cancer diagnosis. Therefore, detection of miRNAs has great significance not only for the studies of miRNAs'biological functions but also for clinical diagnosis.In this paper, by using miRNAs to initiate loop-mediated isothermal amplification reaction (LAMP), we report a simple, rapid and specific miRNA assay. As low as 1.0 amol miRNA can be quantitatively detected with the LAMP-based method, and the dynamic range spans over 6 orders of magnitude. Due to the short length, similar nucleotide sequences, and low expression levels of miRNAs, it's difficult to discriminate each other between the miRNA family members. The proposed miRNA assay can be characterized with high selectivity to discriminate one base difference among miRNA sequences. Furthermore, the proposed miRNA assay has been applied to determine the amount of let-7a in human lung total RNA samples and the amount of let-7a in the 10 ng total RNA samples was estimated to be 50 amol. The LAMP-based miRNA assay can be processed with one type of DNA polymerase and DNA template and primers without any modification with one-step operation. The products are monitored by using SYBR Green ? fluorescent dye, which should reduce the cost of experiments and simplify the experimental procedure. Therefore, the microRNA assay should contribute to future advances in research by using microRNAs as the targets.