Sequential Comparative Hybridizations Analyzed by Computerized Image Processing Can Identify and Quantitate Regulated RNAs

ABSTRACT A method to analyze shifts in the relative abundance of many specific RNAs following any stimulus is presented. Hybridizations of two complex "total" cDNA probes (from the pre- and poststimulus states) to each member of a cDNA library are quantitatively analyzed and mathematically compared by using computer-assisted image processing and statistical analysis of sequential filter hybridizations. Experiments indicate that shifts in abundance between two states can be identified and reproducibly quantitated without purified probes. Direct isolation of recombinant cDNA colonies containing inserts corresponding to regulated RNAs is thus possible. The use of this system is demonstrated for partial analysis of the response in vivo of rat liver to glucocorticoids. Application to other biological systems in which a shift between two states occurs is discussed.