Pyrosequencing for accurate imprinted allele expression analysis.
J Cell Biochem
; 116(7): 1165-70, 2015 Jul.
Article
em En
| MEDLINE
| ID: mdl-25581900
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.
Palavras-chave
Texto completo:
1
Bases de dados:
MEDLINE
Assunto principal:
Fator de Crescimento Insulin-Like II
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Análise de Sequência de DNA
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Impressão Genômica
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Sequenciamento de Nucleotídeos em Larga Escala
Tipo de estudo:
Diagnostic_studies
Limite:
Animals
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Humans
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Male
Idioma:
En
Revista:
J Cell Biochem
Ano de publicação:
2015
Tipo de documento:
Article