# Protocol for analysis of miRNAs in human melanoma cells

## TL;DR

This paper provides a detailed protocol for analyzing miRNAs in human melanoma cells to better understand their role in cancer progression.

## Contribution

The paper introduces a novel protocol for co-detection of miRNAs and mRNAs in melanoma cells using specific staining and imaging techniques.

## Key findings

- Two cell plating approaches are described for preparing melanoma cells for miRNA analysis.
- The RNAscope Plus smRNA-RNA HD assay is used for cell pretreatment and staining.
- Quantitative analysis methods for miRNA expression and co-localization are detailed.

## Abstract

MicroRNAs (miRNAs) significantly contribute to melanoma plasticity, a key factor in metastasis and therapy resistance. Here, we provide a protocol for analysis of miRNAs in human melanoma cells. We describe two approaches for plating cells, either using cell chambers or attaching cells to microscopy slides by means of centrifugation. We first focus on cell pretreatment and staining procedures using the RNAscope Plus small RNA-RNA high-definition (RNAscope Plus smRNA-RNA HD) assay. We then detail the quantitative analysis of miRNA expression and co-localization.

•Steps for co-detection of microRNAs and mRNAs in melanoma cells•Instructions for preparing adherent and suspension cells for microRNA/mRNA co-staining•Guide to image acquisition, segmentation, and analysis using Vectra Polaris and QuPath

Steps for co-detection of microRNAs and mRNAs in melanoma cells

Instructions for preparing adherent and suspension cells for microRNA/mRNA co-staining

Guide to image acquisition, segmentation, and analysis using Vectra Polaris and QuPath

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

MicroRNAs (miRNAs) significantly contribute to melanoma plasticity, a key factor in metastasis and therapy resistance. Here, we provide a protocol for analysis of miRNAs in human melanoma cells. We describe two approaches for plating cells, either using cell chambers or attaching cells to microscopy slides by means of centrifugation. We first focus on cell pretreatment and staining procedures using the RNAscope Plus small RNA-RNA high-definition (RNAscope Plus smRNA-RNA HD) assay. We then detail the quantitative analysis of miRNA expression and co-localization.