In situ Hybridization (ISH) and Quantum Dots (QD) of miRNAs

Sajni Josson; Murali Gururajan; Leland W K Chung

doi:10.21769/BioProtoc.2138

In situ Hybridization (ISH) and Quantum Dots (QD) of miRNAs

Bio Protoc. 2017 Feb 20;7(4):e2138. doi: 10.21769/BioProtoc.2138.

Authors

Sajni Josson^{1

2}, Murali Gururajan^{1

3}, Leland W K Chung¹

Affiliations

¹ Uro-Oncology Research Program, Department of Medicine, Samuel Oschin Comprehensive Cancer Institute, Cedars-Sinai Medical Center, Los Angeles, USA.
² Present address: Neostrata Inc, Princeton, USA.
³ Present address: Bristol-Myers Squibb Inc, Princeton, USA.

Abstract

miRNA are short non-coding RNA which inhibit translation of mRNA. miRNA regulate several cellular processes. Certain miRNA are known to induce oncogenesis. miRNA can be measured by real-time PCR and be imaged using a combination of in situ hybridization (ISH) and quantum dots (QD). The advantage of using quantum dots is that several miRNA can be simultaneously measured using multiplexed QD. Additionally, miRNA can be visualized in different regions of the tissue. Since miRNA are biomarkers of various disease states, miRNA can be visualized and quantitated in tissue sections for diagnostic and prognostic purposes. Here we describe ISH-QD analysis of tissue sections. Tissue sections from xenografts or clinical specimens are used. These are deparaffinized, treated with Proteinase K and hybridized with a biotin-probe to specific to the miRNA. The in situ hybridization is performed by labeling the biotin-probes and followed by labeling with streptavidin tagged quantum dots. Image acquisition of the quantum dots is performed and analyzed for the miRNA expression levels. Combining ISH and QD gives a powerful tool to detect miRNA in different cells of the tissue.

Keywords: Biomarkers; Cancer; Cancer associated stroma; Cellular compartments; Multiplexed quantum dots; Tissue staining; in situ hybridization; miRNA.

Abstract

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