Enzymatic Method

Abstract

The enzymatic miRNA detection method is an enzyme-linked immunosorbent assay (ELISA) procedure specifically modified for the detection and quantification of miRNAs. The technology was developed by Mora and Getts [(Biotechniques 41:420–424, 2006); Genisphere, Inc., Hatfield, PA, USA] based on a previous study designed for miRNA profiling with microarray technology reported by Goff et al. (RNA Biol 2:e9–e16, 2005). The technology consists of three parts: miRNA probes, labeled miRNAs, and horseradish peroxidase (HRP)-conjugated DNA dendrimers as detection molecules for signal amplification. The method offers several advantages for miRNA analysis over microarrays: (1) it takes shorter experimental time, (2) it is less expensive, and (3) it has high sample throughput for studying the expression of the same miRNA in many different samples. The disadvantages of the method are: (1) a given experiment can only investigate as many sequences as the number of wells analyzed and (2) in most cases, each well requires approximately 3 ng labeled LMW RNA to generate signal/noise ratio  > 3. Moreover, the study of some miRNAs, such as let-7f might require more than 30 ng/well. The technique has been validated with tissue-specific expression of miR-1 in heart tissue, miR-122 in liver, and miR-124a in brain (Biotechniques 41:420–424, 2006).