Signal Quantification

Many investigators regard the use of Southern and Northern blot technologies as qualitative tools. They look for the presence or absence of signals or, at best, at faint, strong, and intense signal differences. Older methodologies to quantify autoradiographic images as well as the more recent imaging technologies previously discussed are available. The older methodology, quantitative densitometric scanning, involves the determination of the optical density of autoradiographic signals on X-ray film using a laser scanner. This technique requires rigorous attention to film exposure conditions and the simultaneous use of a series of known standards to determine the linear (dynamic) range of the assay. The X-ray film should be preflashed to an optical density of approximately 0.05 with a shielded light source before autoradiography to improve the dynamic range of the assay at low signal intensities. This technique, under the most ideal conditions and after multiple autoradiographic exposures and densitometer runs, is capable of a dynamic range of only 1.5-2 orders of magnitude. Despite these limitations, laser densitometers are affordable ($10,000 to $15,000) for many laboratories.

Innovations in image processing of radioactive samples involve the use of direct detection of radioactive emissions or the detection of radioactive energy stored in phosphor screens previously exposed to radioactive samples. These technologies are capable of quantifying radioactive samples with dynamic ranges of 3-5 orders of magnitude without the cumbersome constraints of laser densitometry. The price of these instruments ($50,000 to $100,000) puts this technology out of the reach of many investigators.

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