Multiplexed Detection and Analysis of Low-Abundance Long Noncoding RNA Using RNAscope™ in Cultured Cells

Julia Jones; Heather Zecchini; Sankari Nagarajan

doi:10.1007/978-1-0716-0623-0_7

Multiplexed Detection and Analysis of Low-Abundance Long Noncoding RNA Using RNAscope™ in Cultured Cells

Methods Mol Biol. 2020:2148:111-125. doi: 10.1007/978-1-0716-0623-0_7.

Authors

Julia Jones¹, Heather Zecchini², Sankari Nagarajan³

Affiliations

¹ Cancer Research UK, University of Cambridge, Cambridge, UK. julia.jones@cruk.cam.ac.uk.
² Light Microscopy Core Facility, Cancer Research UK/Cambridge Institute, University of Cambridge, Li Ka Shing Centre, Cambridge, UK.
³ Nuclear Hormone Receptor Laboratory, Cancer Research UK/Cambridge Institute, University of Cambridge, Li Ka Shing Centre, Cambridge, UK.

PMID: 32394378
DOI: 10.1007/978-1-0716-0623-0_7

Abstract

Detecting low-abundance long noncoding RNAs (lncRNAs) is extremely difficult due to their expression levels. Deeper sequencing with extensive protocols is required to detect these RNAs and high-throughput screens to examine the regulation of these RNAs are challenging. This protocol provides a multiplexed and robust method of detecting low-abundance RNAs, with improved signal-to-noise ratio using RNAscope-based RNA-FISH which utilizes a series of amplification steps. We have validated this protocol for investigating the regulation of low-abundance lncRNAs, which would be ideal for in vitro screening in 96-well plates.

Keywords: Multiplex RNA-FISH; Operetta; RNA-FISH; RNAscope; lncRNAs.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Cells, Cultured
High-Throughput Screening Assays / methods*
Humans
Molecular Biology / methods*
RNA, Long Noncoding / genetics
RNA, Long Noncoding / isolation & purification*

Substances

RNA, Long Noncoding