A simple and rapid new method for miRNA detection

　　Background: MicroRNAs (miRNAs) are a class of non-coding small RNAs (~22 nt) that have been discovered in recent years and are highly conserved in evolution. As key post-transcriptional gene expression regulators, they can participate in almost all physiological and pathological processes, revolutionizing our traditional understanding of gene expression regulation. The detection of miRNAs is the basis for their functional research, but the characteristics of small fragments make their detection very difficult.

　　PCR and Northern blot are the two most widely used methods for detecting single miRNA. The PCR method has high sensitivity, but the concerns about false positives and the difficulty of primer design limit its use. Northern blot is a very effective detection tool, but the traditional solid phase hybridization technology is complicated, time-consuming and inconvenient to operate. The newly emerging liquid phase hybridization technology has a fast detection speed, but requires a specific fluorescence signal detection instrument .

　　Method improvement: Here we report a new miRNA detection method: liquid hybridization chromogenic assay (LHCD).

　　This method involves liquid phase hybridization of the extracted and purified small RNA with a 5'-end biotin-labeled DNA probe, followed by digestion of excess probe with exonuclease, and then spotting the hybridization solution on a nylon membrane, followed by ABC incubation, and finally BCIP/NBP, DAB or ECL color development.

　　LHCD can distinguish the difference of one base between members of the miRNA family, and its sensitivity can reach 2.5fmol, and the whole process takes less than 3 hours. This method integrates two technologies, rapid liquid hybridization and avidin-biotin signal amplification system (ABC), which not only realizes the simple, rapid and highly sensitive detection of miRNA, but also eliminates the expensive signal detector with its visual signal inspection. In addition, the simple operation process of this method makes it easy to learn and convenient to use.