A RNAscope whole mount approach that can be combined with immunofluorescence to quantify differential distribution of mRNA

Jennifer Kersigo; Ning Pan; Joseph D Lederman; Snehajyoti Chatterjee; Ted Abel; Gabriela Pavlinkova; Immaculada Silos-Santiago; Bernd Fritzsch

doi:10.1007/s00441-018-2864-4

Back

A RNAscope whole mount approach that can be combined with immunofluorescence to quantify differential distribution of mRNA

Journal article

Peer reviewed

A RNAscope whole mount approach that can be combined with immunofluorescence to quantify differential distribution of mRNA

Jennifer Kersigo, Ning Pan, Joseph D Lederman, Snehajyoti Chatterjee, Ted Abel, Gabriela Pavlinkova, Immaculada Silos-Santiago and Bernd Fritzsch

Cell and tissue research, Vol.374(2), pp.251-262

11/2018

DOI: 10.1007/s00441-018-2864-4

PMCID: PMC6878655

PMID: 29974252

Files and links (1)

url

https://www.ncbi.nlm.nih.gov/pmc/articles/6878655View

Open Access

Abstract

RNAscope® technology provided by Advanced Cell Diagnostics (ACD) allows the detection and evaluation of coinciding mRNA expression profiles in the same or adjacent cells in unprecedented quantitative detail using multicolor fluorescent in situ hybridization (FISH). While already extensively used in thinly sectioned material of various pathological tissues and, to a lesser extent, in some whole mounts, we provide here a detailed approach to use the fluorescent RNAscope method in the mouse inner ear and thick brain sections by modifying and adapting existing techniques of whole mount fluorescent in situ hybridization (WH-FISH). We show that RNAscope WH-FISH can be used to quantify local variation in overlaying mRNA expression intensity, such as neurotrophin receptors along the length of the mouse cochlea. We also show how RNAscope WH-FISH can be combined with immunofluorescence (IF) of some epitopes that remain after proteinase digestion and, to some extent, with fluorescent protein markers such as tdTomato. Our WH-FISH technique provides an approach to detect cell-specific quantitative differences in developing and mature adjacent cells, an emerging issue revealed by improved cellular expression profiling. Further, the presented technique may be useful in validating single-cell RNAseq data on expression profiles in a range of tissue known or suspected to have locally variable mRNA expression levels.

Fluorescent Antibody Technique - methods

Mice, Inbred C57BL

RNA, Messenger - genetics

Gene Expression Regulation

In Situ Hybridization, Fluorescence

Receptor, trkB - genetics

RNA, Messenger - metabolism

Animals

Receptor, trkC - genetics

Neurotrophin 3 - metabolism

Cochlea - metabolism

Mice, Inbred BALB C

Imaging, Three-Dimensional

Receptor, trkB - metabolism

Receptor, trkC - metabolism

Details

Title: Subtitle: A RNAscope whole mount approach that can be combined with immunofluorescence to quantify differential distribution of mRNA
Creators: Jennifer Kersigo - Department of Biology, University of Iowa, Iowa City, IA, 52242, USA
Ning Pan - Decibel Therapeutics, Boston, MA, USA
Joseph D Lederman - Iowa Neuroscience Institute, University of Iowa, Iowa City, IA, USA
Snehajyoti Chatterjee - Iowa Neuroscience Institute, University of Iowa, Iowa City, IA, USA
Ted Abel - Iowa Neuroscience Institute, University of Iowa, Iowa City, IA, USA
Gabriela Pavlinkova - Institute of Biotechnology CAS, Vestec, Czechia
Immaculada Silos-Santiago - Decibel Therapeutics, Boston, MA, USA
Bernd Fritzsch - Department of Biology, University of Iowa, Iowa City, IA, 52242, USA. bernd-fritzsch@uiowa.edu
Resource Type: Journal article
Publication Details: Cell and tissue research, Vol.374(2), pp.251-262
DOI: 10.1007/s00441-018-2864-4
PMID: 29974252
PMCID: PMC6878655
NLM abbreviation: Cell Tissue Res
ISSN: 0302-766X
eISSN: 1432-0878
Publisher: Germany
Grant note: R01 DC005590 / NIH/NIDCD
Language: English
Date published: 11/2018
Academic Unit: Molecular Physiology and Biophysics; Psychiatry; Psychological and Brain Sciences; Iowa Neuroscience Institute; Biology; Craniofacial Anomalies Research Center; Neuroscience and Pharmacology; Biochemistry and Molecular Biology
Record Identifier: 9984070160802771

Metrics

34 Record Views

37 Times Cited - Web of Science