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 33P or 35S 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 33P-labelled oligonucleotides together with 14C-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 33P-standards for each experiment was replaced by co-exposition and calibration of long-lived 14C-standards together with 33P-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 α-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.