Calibration of densitometry in radio-isotopic in situ hybridization

Densitometry on autoradiographs of sections processed for in situ hybridization provides a direct measure for the in situ quantification of mRNA. Gelatin spots, containing different concentrations of the radioisotope, and processed in parallel with the tissue sections, can be used as a sensitive model to calibrate the densitometric measurements. The shape of the gelatin spots was shown to be circular with a parabolic crosssectional profile. This simple shape allows the subdivision of the spot into a series of concentric rings, which enables an unbiased measurement of the optical density - radioactivity relation. This spot measurement is also applicable to DNA arrays spotted on glass or membranes. A new model, explaining the optical density of autoradiographs, was derived and fitted to the calibration points. The use of this calibration method is crucial for the correct interpretation of autoradiographs.