Quantification of miRNA abundance in single cells using locked nucleic acid-FISH and enzyme-labeled fluorescence

The ability to quantify miRNA abundance at the single-cell level and image its spatial distribution could lead to unique insight into the biological roles of miRNAs and miRNA-associated gene regulatory networks. This protocol describes a method for quantitatively imaging miRNAs in single cells using fluorescence in situ hybridization (FISH). The method combines the unique miRNA recognition properties of locked nucleic acid (LNA) with the signal amplification technology known as enzyme-labeled fluorescence (ELF). Although both approaches have previously been shown to increase detection specificity and/or sensitivity in FISH, combining these techniques into one protocol allows for single molecule detection. Specifically, individual miRNAs are identified as bright, photostable fluorescent spots. The dynamic range was found to span over three orders of magnitude and the average miRNA copy number per cell was within 17.5% of measurements acquired by quantitative RT-PCR.