title: Pyrosequencing for accurate imprinted allele expression analysis.
abstract: Genomic imprinting is an epigenetic mechanism that restricts gene expression to one inherited allele. Improper maintenance of imprinting has been implicated in a number of human diseases and developmental syndromes. Assays are needed that can quantify the contribution of each paternal allele to a gene expression profile. We have developed a rapid, sensitive quantitative assay for the measurement of individual allelic ratios termed Pyrosequencing for Imprinted Expression (PIE). Advantages of PIE over other approaches include shorter experimental time, decreased labor, avoiding the need for restriction endonuclease enzymes at polymorphic sites, and prevent heteroduplex formation which is problematic in quantitative PCR-based methods. We demonstrate the improved sensitivity of PIE including the ability to detect differences in allelic expression down to 1%. The assay is capable of measuring genomic heterozygosity as well as imprinting in a single run. PIE is applied to determine the status of Insulin-like Growth Factor-2 (IGF2) imprinting in human and mouse tissues.
authors.affiliation: Department of Urology, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin.
authors.forename: Bing
authors.initials: B
authors.lastname: Yang
authors.affiliation: Department of Urology, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin.
authors.forename: Nathan
authors.initials: N
authors.lastname: Damaschke
authors.affiliation: Department of Urology, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin.
authors.forename: Tianyu
authors.initials: T
authors.lastname: Yao
authors.affiliation: Department of Urology, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin.
authors.forename: Johnathon
authors.initials: J
authors.lastname: McCormick
authors.affiliation: Department of Urology, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin.
authors.forename: Jennifer
authors.initials: J
authors.lastname: Wagner
authors.affiliation: Department of Urology, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin.|University of Wisconsin Carbone Comprehensive Cancer Center, Madison, Wisconsin.|Environmental and Molecular Toxicology, University of Wisconsin, Madison, Wisconsin.
authors.forename: David
authors.initials: D
authors.lastname: Jarrard
mesh_terms: D000483:Alleles; D000818:Animals; D020869:Gene Expression Profiling; D018392:Genomic Imprinting; D006579:Heterozygote; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D007335:Insulin-Like Growth Factor II / Q000235:genetics*; D008297:Male; D051379:Mice; D020641:Polymorphism, Single Nucleotide; D011467:Prostate / Q000378:metabolism; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA / Q000379:methods*
chemical_list: D007335:Insulin-Like Growth Factor II
keywords: EPIGENETIC; IGF2; IMPRINTING; INTRON-CROSSING PRIMER; PYROSEQUENCING