MicroRNA Amplification and Recognition through Locked-nucleic-acid In situ Hybridization as A Novel Detection and Quantification Method

When using traditional probe-based in situ hybridization (ISH) imaging detection methods, microRNAs (miRNAs) are difficult to capture due to their size, which ranges from 19 to 25 nucleotides. However, as miRNAs are key epigenetic regulators that contribute to normal physiology and numerous pathological conditions, understanding the spatiotemporal behavior of miRNAs is important to unravel their function at the cellular and subcellular levels. This creates a clear need for adequate detection and visualization techniques. Therefore, we created MicroRNA Amplification and Recognition through Locked-nucleic-acid in situ hybridizatioN (MARLIN), which was developed and optimized utilizing multiplex immunofluorescence staining, and locked nucleic acid (LNA) probes targeted to a miRNA. This method can quantify miRNA with multiplexed protein expression, which allows the quantification of intracellular and extracellular miRNA within the tumor microenvironment (TME) with high sensitivity and reproducibility. The combined use of miRNA detection and protein capture assays can also provide a comprehensive understanding of cellular functions and regulatory mechanisms within that TME.