A NEW QUANTITATIVE FILM AUTORADIOGRAPHIC METHOD OF QUANTIFYING MESSENGER-RNA TRANSCRIPTS FOR IN-SITU HYBRIDIZATION

We developed and tested a novel quantitative method for the quantifica tion of film autoradiographs, involving a mathematical model and a dot -blot-based membrane standard scale. The exponential model introduced here, ROD = p(1)(1 - exp[p(2)x]), appropriately (r(2)>0.999), describe s the relation between relative optical density (ROD) and radioactivit y (x) in the range between 0 and 240 gray scale values (using a 256-gr ay scale level digitizer). By means of this model, standard curves wit h distinct quenching properties can be exactly interconverted, permitt ing the tissue-equivalent calibration of different standard scales. Th e membrane standard scale employed here has several advantages, includ ing the flexible radioactivity range, the facile and rapid preparation technique, and the compact size. The feasibility of the quantificatio n procedure is exemplified by the comparative quantification of multip le calmodulin mRNAs in the rat brain by in situ hybridization with [S- 35]-cRNA probes. The procedure for quantification provides a significa nt 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 autorad iographs produced by other methods.