Multicolor microRNA FISH effectively differentiates tumor types

Abstract

MicroRNAs (miRNAs) are excellent tumor biomarkers because of their cell-type specificity and abundance. However, many miRNA detection methods, such as real-time PCR, obliterate valuable visuospatial information in tissue samples. To enable miRNA visualization in formalin-fixed paraffin-embedded (FFPE) tissues, we developed multicolor miRNA FISH. As a proof of concept, we used this method to differentiate two skin tumors, basal cell carcinoma (BCC) and Merkel cell carcinoma (MCC), with overlapping histologic features but distinct cellular origins. Using sequencing-based miRNA profiling and discriminant analysis, we identified the tumor-specific miRNAs miR-205 and miR-375 in BCC and MCC, respectively. We addressed three major shortcomings in miRNA FISH, identifying optimal conditions for miRNA fixation and ribosomal RNA (rRNA) retention using model compounds and high-pressure liquid chromatography (HPLC) analyses, enhancing signal amplification and detection by increasing probe-hapten linker lengths, and improving probe specificity using shortened probes with minimal rRNA sequence complementarity. We validated our method on 4 BCC and 12 MCC tumors. Amplified miR-205 and miR-375 signals were normalized against directly detectable reference rRNA signals. Tumors were classified using predefined cutoff values, and all were correctly identified in blinded analysis. Our study establishes a reliable miRNA FISH technique for parallel visualization of differentially expressed miRNAs in FFPE tumor tissues.

A whole-mount in situ hybridization method for microRNA detection in Caenorhabditis elegans

“…There is a trade-off between these parameters. The fixation of mouse sectioned tissues with EDC is initially carried out by incubation at 25°C (Pena et al 2009), which is subsequently increased to 50°C to achieve near-complete phosphoramide bond formation in a shorter time (Renwick et al 2013). Cross-linking of miRNAs with EDC for Northern blotting also shows that increasing the reaction temperature up to 60°C improves miRNA detection (Pall et al 2007).…”

“…More cross-linking at higher temperatures must lead to more retention of miRNAs, leading to stronger hybridization signals. However, cross-linking with EDC acts not only on miRNAs but also on proteins to form peptide bonds, which make the proteins resistant to proteinase (Sung et al 2003;Renwick et al 2013). As the surfaces of C. elegans larvae and adults are overlaid with cuticle layers composed of collagens, the layers have to be partially digested by proteinase so that WISH reagents, such as probes, can penetrate inward.…”

MicroRNAs: An adaptive mechanism in the pancreatic β-cell…and beyond?

“…While several sequences including miR-375, miR-7, and miR-184 appear to be enriched in the endocrine pancreas, profiling results from mouse and human tissues have shown that none of these miRNAs are entirely specific to the islet [7] [8]. In the case of miR-375, expression of this miRNA has been reported in numerous tissue types including the pituitary gland, adrenal gland, intestine, lung, and several types of cancer [9] [10]. Similarly, miR-7 and miR-184 have been shown to be highly enriched in the central nervous system and eye, respectively [11].…”

RNA-guided diagnostics and therapeutics for next-generation individualized nanomedicine

“…In this issue of the JCI, Renwick et al (20) report on a method for detection and measurement of microRNAs in clinical samples (20). This work represents a significant technical advance in the development of a reliable, clinical laboratory-compatible RNA FISH methodology for molecular diagnostic applications.…”

“…A notable limitation of the study is that the RNA FISH-based methodology was conclusively validated only for two tumor-specific miRNAs (miR-205 and miR-375) in the small number of clinical samples analyzed to date. In the context of the expressed commitment of this group to explore the clinical utility of RNA FISH-based microRNA analyses in larger collections of clinical samples (20), this study has significant translational implications, because altered expression and activity of miR-205 and miR-375 has been linked to the increased likelihood of developing castration-resistant phenotype of human prostate cancer (13).…”