A new quantitative film autoradiographic method of quantifying mRNA transcripts for in situ hybridization.

ABSTRACT

We developed and tested a novel quantitative method for the quantification of film autoradiographs, involving a mathematical model and a dot-blot-based membrane standard scale. The exponential model introduced here, ROD = p1(1 - exp[p2x]), appropriately (r2>0. 999), describes the relation between relative optical density (ROD) and radioactivity (x) in the range between 0 and 240 gray scale values (using a 256-gray scale level digitizer). By means of this model, standard curves with distinct quenching properties can be exactly interconverted, permitting the tissue-equivalent calibration of different standard scales. The membrane standard scale employed here has several advantages, including the flexible radioactivity range, the facile and rapid preparation technique, and the compact size. The feasibility of the quantification procedure is exemplified by the comparative quantification of multiple calmodulin mRNAs in the rat brain by in situ hybridization with [35S]-cRNA probes. The procedure for quantification provides a significant improvement in that the direct and exact comparison of radiolabeled species, even from different experiments, can be reliably performed. Further, the procedure can be adapted to the quantification of autoradiographs produced by other methods.