Quantification of Cytochrome P450 Gene Expression by RNase Protection Analysis

Many attempts to describe the regulation of mdivldual cytochrome P450 (CYP) gene products in animal tissues have been frustrated by the lack of sensitivity and specificity of the assays used. Most enzymatic assays are hampered by the overlapping substrate specificity of these enzymes, and the generation of high-affinity antibodies that are specific for individual isoforms is not a trivial task. Many CYP isoforms have close relatives, some of which can have as much as 98% identity at the RNA nucleotlde-sequence level, and filter-hybridization techniques using labeled cDNA probes are unable to dlscrlminate between such mRNAs. Although noncoding portions of the cDNAs may be used as probes, as they are more likely to offer regions of divergence within subfamllles, this is not often possible because