MicroRNAs (miRNAs) play important roles in nearly every aspect of biology, including physiological, biochemical, developmental and pathological processes. Therefore, a highly sensitive and accurate method of detection of miRNAs has great potential in research on theory and application, such as the clinical approach to medicine, animal and plant production, as well as stress response. Here, we report a strategic method to detect miRNAs from multicellular organisms, which mainly includes liquid hybridization and solid phase detection (LHSPD); it has been verified in various species and is much more sensitive than traditional biotin-labeled Northern blots. By using this strategy and chemiluminescent detection with digoxigenin (DIG)-labeled or biotin-labeled oligonucleotide probes, as low as 0.01-0.25 fmol [for DIG-CDP Star (disodium2-chloro-5-(4-methoxyspiro{1,2-dioxetane-3,2'-(5'-chloro)tricyclo[3.3.1.13,7]decan}-4-yl)phenyl phosphate) system], 0.005-0.1 fmol (for biotin-CDP Star system), or 0.05-0.5 fmol (for biotin-luminol system) of miRNA can be detected and one-base difference can be distinguished between miRNA sequences. Moreover, LHSPD performed very well in the quantitative analysis of miRNAs, and the whole process can be completed within about 9 h. The strategy of LHSPD provides an effective solution for rapid, accurate, and sensitive detection and quantitative analysis of miRNAs in plants and animals.
Keywords: biotin; digoxigenin; liquid hybridization; microRNA detection; solid phase detection.