title: Pyrosequencing for the rapid and efficient quantification of allele-specific expression.
abstract: We have developed a rapid and sensitive quantitative assay for the measurement of individual allelic ratios. This assay minimizes time and labor, the need for special restriction endonuclease enzymes for polymorphic sites, and avoids heteroduplex formation seen with traditional quantitative PCR-based methods. It has improved sensitivity compared to other methods and is capable of distinguishing 1% differences in allelic expression. This assay, termed Pyrosequencing for Imprinted Expression (PIE), involves the use of an intron-crossing PCR primer to generate the first PCR product. We applied the assay to analyze Insulin-like Growth Factor-2 (IGF2) imprinting in both human and mouse prostate tissues.
authors: Department of Urology; University of Wisconsin School of Medicine and Public Health; Madison, WI USA.
authors: Bing
authors: B
authors: Yang
authors: Department of Urology; University of Wisconsin School of Medicine and Public Health; Madison, WI USA.
authors: Jennifer
authors: J
authors: Wagner
authors: Department of Urology; University of Wisconsin School of Medicine and Public Health; Madison, WI USA.
authors: Tianyu
authors: T
authors: Yao
authors: Department of Urology; University of Wisconsin School of Medicine and Public Health; Madison, WI USA.
authors: Nathan
authors: N
authors: Damaschke
authors: Department of Urology; University of Wisconsin School of Medicine and Public Health; Madison, WI USA; University of Wisconsin Carbone Comprehensive Cancer Center; Madison, WI USA; Environmental and Molecular Toxicology; University of Wisconsin; Madison, WI USA.
authors: David F
authors: DF
authors: Jarrard
mesh_terms: D000483:Alleles; D000818:Animals; D015870:Gene Expression; D018392:Genomic Imprinting; D006801:Humans; D007335:Insulin-Like Growth Factor II / Q000235:genetics* / Q000378:metabolism; D008297:Male; D051379:Mice; D011467:Prostate / Q000378:metabolism; D017422:Sequence Analysis, DNA / Q000379:methods*
chemical_list: D007335:Insulin-Like Growth Factor II
keywords: IGF2; epigenetic; imprinting; intron-crossing primer; pyrosequencing
references: Novartis Found Symp. 2004;262:108-21; discussion 121-4, 265-8
references: 15562825
references: Nat Genet. 2000 Aug;25(4):375-6
references: 10932175
references: C R Biol. 2012 Jun;335(6):398-405
references: 22721561
references: J Biol Chem. 2004 Dec 10;279(50):52218-26
references: 15471867
references: Anal Biochem. 1998 Jun 15;260(1):24-9
references: 9648648
references: Genes Chromosomes Cancer. 2005 Nov;44(3):256-64
references: 16001432
references: Biochem Biophys Res Commun. 1997 Jun 9;235(1):123-9
references: 9196048
references: Folia Histochem Cytobiol. 2012 Jul 05;50(2):171-9
references: 22763974
references: Epigenetics. 2008 Sep;3(5):261-9
references: 18769151
references: Cancer. 1999 Mar 15;85(6):1389-94
references: 10189147
references: Methods Mol Biol. 2009;578:123-33
references: 19768590
references: Cancer Res. 2008 Aug 15;68(16):6797-802
references: 18701505
references: Am J Pathol. 1999 Mar;154(3):635-47
references: 10079240
references: BMC Evol Biol. 2010 Sep 13;10:276
references: 20836842
references: Hum Mol Genet. 1993 Jan;2(1):43-50
references: 8490622
references: Curr Issues Mol Biol. 2009;11(1):1-12
references: 18541926
references: Exp Cell Res. 1999 Apr 10;248(1):18-24
references: 10094809