High-Throughput In Situ Hybridization: Systematical Production of Gene Expression Data and Beyond

Protocol
Part of the Neuromethods book series (NM, volume 99)

Abstract

A plethora of modern-day techniques allows the detailed characterization of the transcriptome on a quantitative level. Analyses, based on techniques such as cDNA microarrays or RNA-seq (whole transcriptome shotgun sequencing), are usually genome wide in scope and readily detect small changes in gene expression levels across different biological samples. However, when it comes to spatial localization of gene expression within the context of complex tissues, traditional methods of in situ hybridization remain unparalleled with regard to their cellular resolution.

Here we review methods that extend classical in situ hybridization protocols and techniques to the special needs of high-throughput (HT) studies and which can be readily scaled up to a genomic level to cover organs or even whole organisms in great detail. Moreover, we discuss suitable HT instrumentation and address postproduction issues typically arising with HT pipelines such as annotation of expression data and database organization.

Key words

Annotation Web databases In situ hybridization Functional genomics Gene expression analysis 

Abbreviations

BLAST

Basic local alignment search tool

BLAT

BLAST-like alignment tool

ISH

In situ hybridization

NR

Nonradioactive

RNA

Ribonucleic acids

LNA

Locked nucleic acids

ROI

Region of interest

h

Hours

min

Minutes

s

Seconds

RT

Room temperature

IT

Information technology

XAMPP

Apache distribution containing MySQL, PHP, and Perl

CNS

Central nervous system

E

Embryonic day

P

Postnatal day

DIG

Digoxigenin

NBT

Nitro blue tetrazolium

BCIP

5-Bromo-4-chloro-3-indolyl-phosphate

FA

Formaldehyde

IAA

Iodoacetamide

Notes

Acknowledgments

We thank Christina Thaller, Dirk Reuter, Benjamin Tetzlaff, and Dr. Murat Yaylaoglu for their assistance in the preparation of this manuscript. We acknowledge the support of the Max Planck Society (L.G. and G.E.).

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Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  1. 1.Department of Genes and BehaviorMax Planck Institute for Biophysical ChemistryGöttingenGermany

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