TY  - JOUR
AU  - Cremer, C.M.
AU  - Cremer, M.
AU  - Lopez Escobar, J.
AU  - Speckmann, E.J.
AU  - Zilles, K.
TI  - Fast, quantitative in situ hybridization of rare mRNAs using 14C-standards and phosphorus imaging
JO  - Journal of neuroscience methods
VL  - 185
SN  - 0165-0270
CY  - Amsterdam [u.a.]
PB  - Elsevier Science
M1  - PreJuSER-7022
SP  - 56 - 61
PY  - 2009
N1  - Record converted from VDB: 12.11.2012
AB  - 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.
KW  - Animals
KW  - Cacodylic Acid: toxicity
KW  - Carbon Radioisotopes: chemistry
KW  - Hippocampus: drug effects
KW  - Hippocampus: metabolism
KW  - Hippocampus: physiopathology
KW  - Image Processing, Computer-Assisted: instrumentation
KW  - Image Processing, Computer-Assisted: methods
KW  - In Situ Hybridization: methods
KW  - Male
KW  - Neurochemistry: methods
KW  - Oligonucleotides: chemistry
KW  - Phosphorus Radioisotopes: chemistry
KW  - RNA, Messenger: analysis
KW  - RNA, Messenger: metabolism
KW  - Rats
KW  - Rats, Wistar
KW  - Receptors, AMPA: genetics
KW  - Reference Standards
KW  - Time Factors
KW  - Carbon Radioisotopes (NLM Chemicals)
KW  - Oligonucleotides (NLM Chemicals)
KW  - Phosphorus Radioisotopes (NLM Chemicals)
KW  - RNA, Messenger (NLM Chemicals)
KW  - Receptors, AMPA (NLM Chemicals)
KW  - glutamate receptor ionotropic, AMPA 1 (NLM Chemicals)
KW  - glutamate receptor ionotropic, AMPA 2 (NLM Chemicals)
KW  - Cacodylic Acid (NLM Chemicals)
KW  - J (WoSType)
LB  - PUB:(DE-HGF)16
C6  - pmid:19761793
UR  - <Go to ISI:>//WOS:000272473000008
DO  - DOI:10.1016/j.jneumeth.2009.09.010
UR  - https://juser.fz-juelich.de/record/7022
ER  -