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.
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Notes
- 1.
Metadata include sample identity, riboprobe identity, probe sequence, information on particular protocols used, laboratory staff names, dates of the various procedures carried out, and textual annotation of gene expression patterns.
- 2.
The copper base has an engraved cross hair that can be aligned to an orthogonal grid attached to one of the eye pieces of a dissecting microscope. This alignment is performed prior to adding OCT and specimen. Before removing the chamber, mark its position with flat corner iron brace fastened to the microscope base. Once filled with OCT and specimen, place the chamber back into its original position onto the microscope base and use the grid of the microscope for specimen orientation. Since the sidewalls of the chamber are translucent, the orientation in remaining planes can readily be achieved by visual inspection. A blunt preparation needle should be used to manipulate the specimen.
- 3.
Basic Tm calculation: Tmcalc = 64.9 °C + 41 °C × (number of Gs and Cs in the primer − 16.4)/length of primer. However, a precise optimum annealing temperature must be determined empirically.
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
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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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Geffers, L., Eichele, G. (2015). High-Throughput In Situ Hybridization: Systematical Production of Gene Expression Data and Beyond. In: Hauptmann, G. (eds) In Situ Hybridization Methods. Neuromethods, vol 99. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2303-8_11
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DOI: https://doi.org/10.1007/978-1-4939-2303-8_11
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