Detecting miRNAs by liquid hybridization and color development

Abstract

Currently, two methods, PCR and Northern blot, are widely used to detect individual microRNAs (miRNA). Although PCR is highly sensitive, false positives and difficulties of primer design discourage its use. While a Northern blot is an effective tool, traditional Northern blot protocols are complicated, time-consuming, and usually inconvenient for users. Liquid Northern blot methods are rapid but require instruments for detection of fluorescent signals. Here, we describe an alternative protocol, liquid hybridization and color development (LHCD), based on the rapidity of liquid hybridization and the signal amplification of avidin-biotin complex (ABC) for detection. LHCD can distinguish a one-nucleotide difference within a miRNA family and allow for the sensitive detection of 2.5 fmol of miRNAs. Furthermore, LHCD is not only simple and rapid, but detection is visual and so it does not require expensive equipment. LHCD is easy to learn and convenient for miRNA analyses.