Exposure of @entity35 to peroxisome proliferators induces members of the cytochrome P450 4A (CYP4A) family. In @entity35 , the CYP4A family consists of four related genes, @entity48194 , @entity56433 , @entity61666 , and @entity70036 . We are specifically interested in examining @entity48194 , @entity56433 , and @entity61666 , each of which is expressed in a tissue-dependent and sex-dependent manner. While @entity48194 is sufficiently different from the other two members to enable relatively easy specific quantitation, the close similarity between @entity56433 and @entity61666 makes quantitative discrimination difficult. We have combined a fluorescent real-time PCR assay (TaqMan) with the sequence-specific mismatch amplification mutation assay (MAMA) to allow us to carry out specific quantitation of all three members of this family. The assay is designed such that a single fluorescent TaqMan(R) probe binds to all three gene products, while specificity is conferred by sequence-specific primers. This specific MAMA technique takes advantage of the ability of Taq polymerase to distinguish between the two cDNAs based on mismatches at the 3' end of a PCR primer. In the 84-base PCR product used for this assay, there is only a single-base difference between @entity56433 and @entity61666 . Despite this similarity, there is at least a 1000-fold discrimination between the two sequences, using @entity56433 or @entity61666 specific standards. Analysis of @entity35 liver RNA from both sexes demonstrates that this discrimination is also achieved in complex RNA mixtures. This technique should be broadly applicable to other areas of research such as allelic discrimination, detecting mutational hotspots in @entity5 , and discrimination among closely related members of other gene families.

Use of real-time gene-specific polymerase chain reaction to measure RNA expression of three family members of XXXX cytochrome P450 4A.

@entity56433 :: ('24306(Tax:10116)', 'Gene') :: ['CYP4A2'] @entity70036 :: ('266674(Tax:10116)', 'Gene') :: ['CYP4A8'] @entity5 :: ('MESH:D009369', 'Disease') :: ['tumors'] @entity48194 :: ('50549(Tax:10116)', 'Gene') :: ['CYP4A1'] @entity35 :: ('10116', 'Species') :: ['rat', 'rats'] @entity61666 :: ('298423(Tax:10116)', 'Gene') :: ['CYP4A3']
@entity35 :: (10116,Species) :: ['rat']
