Fast, quantitative in situ hybridization of rare mRNAs using 14C-standards and phosphorus imaging.
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.
Cremer
Christian M
CM
Institute for Neuroscience and Medicine (INM-2), Research Center Jülich, Jülich, Germany. c.cremer@fz-juelich.de
Cremer
Markus
M
Escobar
Jennifer Lopez
JL
Speckmann
Erwin-J
EJ
Zilles
Karl
K
D000818:Animals; D002101:Cacodylic Acid / Q000633:toxicity; D002250:Carbon Radioisotopes / Q000737:chemistry*; D006624:Hippocampus / Q000187:drug effects / Q000378:metabolism / Q000503:physiopathology; D007091:Image Processing, Computer-Assisted / Q000295:instrumentation / Q000379:methods; D017403:In Situ Hybridization / Q000379:methods*; D008297:Male; D009448:Neurochemistry / Q000379:methods; D009841:Oligonucleotides / Q000737:chemistry*; D010761:Phosphorus Radioisotopes / Q000737:chemistry*; D012333:RNA, Messenger / Q000032:analysis* / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D018091:Receptors, AMPA / Q000235:genetics; D012015:Reference Standards; D013997:Time Factors
D002101:Cacodylic Acid; D002250:Carbon Radioisotopes; D009841:Oligonucleotides; D010761:Phosphorus Radioisotopes; D012333:RNA, Messenger; D018091:Receptors, AMPA; C104722:glutamate receptor ionotropic, AMPA 2