Electrocatalytic Moiety Labeling Technique for High-Sensitivity miRNA Expression Analysis

Abstract

Chemical labeling of miRNAs for electrochemical assay has become a useful approach for miRNA detection. It is believed that due to the extremely small size of miRNAs, direct labeling on miRNAs may be more advantageous. Recently, Gao and Yu from the Institute of Microelectronics (Singapore) presented a novel labeling procedure that utilizes a chemical ligation to directly label miRNA with a redox active and catalytic moiety (Biosens Bioelectron 22:933–940, 2007). The miRNA is labeled in the total RNA mixture in a one-step non-enzymatic reaction under very mild conditions with a redox active and electrocatalytic moiety, Ru(PD)₂Cl₂ (PD = 1,10-phenanthroline-5,6-dione), through coordinative bonds with purine bases in the miRNA molecule. The excellent electrocatalytic activity of the Ru(PD)₂Cl₂ towards the oxidation of hydrazine makes it possible to conduct ultrasensitive miRNA detection. Under optimized experimental conditions, the assay allows the detection of miRNAs in the range of 0.50–400 pM with a detection limit of 0.20 pM in 2.5 microl (0.50 amole). miRNA quantitation is, therefore, performed in as little as 10 ng of total RNA, providing a handy platform for miRNA expression analysis. The amplification from the electrocatalytic oxidation of hydrazine greatly enhances the detectability of the assay, thereby lowering the detection limit to 0.2 pM. The electrochemical miRNA assay described here is rapid, ultrasensitive, non-radioactive, and is able to directly detect miRNA without involving biological ligation. The method allows us to identify miRNAs with less than two-fold difference in expression levels under two conditions.