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@ARTICLE{Cremer:7022,
author = {Cremer, C.M. and Cremer, M. and Lopez Escobar, J. and
Speckmann, E.J. and Zilles, K.},
title = {{F}ast, quantitative in situ hybridization of rare m{RNA}s
using 14{C}-standards and phosphorus imaging},
journal = {Journal of neuroscience methods},
volume = {185},
issn = {0165-0270},
address = {Amsterdam [u.a.]},
publisher = {Elsevier Science},
reportid = {PreJuSER-7022},
pages = {56 - 61},
year = {2009},
note = {Record converted from VDB: 12.11.2012},
abstract = {The use of radiolabelled probes for in situ hybridization
(ISH) bears the advantage of high sensitivity and
quantifiability. The crucial disadvantages are laborious
hybridization protocols, exposition of hybridized sections
to film for up to several weeks and the time consuming need
to prepare tissue standards with relatively short-lived
isotopes like (33)P or (35)S for each experiment. The
quantification of rare mRNAs like those encoding for
subunits of neurotransmitter receptors is therefore a
challenge in ISH. Here, we describe a method for fast,
quantitative in situ hybridization (qISH) of mRNAs using
(33)P-labelled oligonucleotides together with (14)C-polymer
standards (Microscales, Amersham Biosciences) and a
phosphorus imaging system (BAS 5000 BioImage Analyzer,
Raytest-Fuji). It enables a complete analysis of rare mRNAs
by ISH. The preparation of short-lived (33)P-standards for
each experiment was replaced by co-exposition and
calibration of long-lived (14)C-standards together with
(33)P-labelled brain paste standards. The use of a
phosphorus imaging system allowed a reduction of exposition
time following hybridization from several weeks to a few
hours or days. We used this approach as an example for
applications to quantify the expression of GluR1 and GluR2
subunit mRNAs of the
alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid
(AMPA) receptor in the hippocampus of untreated rats, and
after intraperitoneal application of the organo-arsenic
compound dimethyl arsenic acid.},
keywords = {Animals / Cacodylic Acid: toxicity / Carbon Radioisotopes:
chemistry / Hippocampus: drug effects / Hippocampus:
metabolism / Hippocampus: physiopathology / Image
Processing, Computer-Assisted: instrumentation / Image
Processing, Computer-Assisted: methods / In Situ
Hybridization: methods / Male / Neurochemistry: methods /
Oligonucleotides: chemistry / Phosphorus Radioisotopes:
chemistry / RNA, Messenger: analysis / RNA, Messenger:
metabolism / Rats / Rats, Wistar / Receptors, AMPA: genetics
/ Reference Standards / Time Factors / Carbon Radioisotopes
(NLM Chemicals) / Oligonucleotides (NLM Chemicals) /
Phosphorus Radioisotopes (NLM Chemicals) / RNA, Messenger
(NLM Chemicals) / Receptors, AMPA (NLM Chemicals) /
glutamate receptor ionotropic, AMPA 1 (NLM Chemicals) /
glutamate receptor ionotropic, AMPA 2 (NLM Chemicals) /
Cacodylic Acid (NLM Chemicals) / J (WoSType)},
cin = {INM-2 / JARA-BRAIN},
ddc = {610},
cid = {I:(DE-Juel1)INM-2-20090406 / $I:(DE-82)080010_20140620$},
pnm = {Funktion und Dysfunktion des Nervensystems},
pid = {G:(DE-Juel1)FUEK409},
shelfmark = {Biochemical Research Methods / Neurosciences},
typ = {PUB:(DE-HGF)16},
pubmed = {pmid:19761793},
UT = {WOS:000272473000008},
doi = {10.1016/j.jneumeth.2009.09.010},
url = {https://juser.fz-juelich.de/record/7022},
}