RNA Detection

Chapter 13

In Situ Detection of MicroRNA Expression

with RNAscope Probes

Viravuth P. Yin

Abstract

Elucidating the spatial resolution of gene transcripts provides important insight into potential gene
function. MicroRNAs are short, singled-stranded noncoding RNAs that control gene expression through
base-pair complementarity with target mRNAs in the 3^0 untranslated region (UTR) and inhibiting protein
expression. However, given their small size of ~22- to 24-nt and low expression levels, standard in situ
hybridization detection methods are not amendable for microRNA spatial resolution. Here, I describe a
technique that employs RNAscope probe design and propriety amplification technology that provides
simultaneous single molecule detection of individual microRNA and its target gene. This method allows
for rapid and sensitive detection of noncoding RNA transcripts in frozen tissue sections.

Key wordsMicroRNAs, In situ hybridization, RNA detection, Immunohistochemistry, RNAscope

1 Introduction

In situ nucleic acid hybridization (ISH) is an essential technique in

biology to define the cellular distribution of nucleic acids within

frozen tissues. Typically, a labeled segment of complementary DNA

or RNA nucleic acid strand (probe) is used to localize a nucleic acid

sequence of interest. First described in the 1950s by Gall and

colleagues in studies ofDrosophila salivary glands andXenopus

oocytes, ISH has become an important first step to defining gene

function [1–3].

With advances in technology and popularity in transcriptome

sequencing, our understanding of RNA biology has greatly

expanded, and now includes both coding and noncoding classifica-

tion. One subclass of noncoding RNAs are small, single-stranded

regulatory RNAs termed microRNAs. Two different enzyme com-

plexes are responsible for processing microRNA transcripts into

their functional single-stranded unit of ~22-nt. In the nucleus, an

RNAse III-like enzyme, Drosha, cleaves microRNA transcripts to

produce a ~65-nt stem-loop structure. Once transported into the

Imre Gaspar (ed.),RNA Detection: Methods and Protocols, Methods in Molecular Biology,
vol. 1649, DOI 10.1007/978-1-4939-7213-5_13,©Springer Science+Business Media LLC 2018

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