Pyrosequencing for the rapid and efficient quantification of allele-specific expression

Yang, Bing; Wagner, Jennifer; Yao, Tianyu; Damaschke, Nathan; Jarrard, David F.

doi:10.4161/epi.25892

Cited by 7 publications

(3 citation statements)

References 18 publications

(16 reference statements)

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“…To examine methylation across whole regions quantitatively, bisulfite pyrosequencing was performed as we have described [51]. Bisulfite-modified DNA was then amplified using PCR in preparation for pyrosequencing, with either the forward or reverse primer biotinylated.…”

Section: Methylation Analyses Including Combined Bisulfite Restrictiomentioning

confidence: 99%

CTCF loss mediates unique DNA hypermethylation landscapes in human cancers

et al. 2020

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Background: The chromatin insulator CCCTC-binding factor (CTCF) displays tissue-specific DNA binding sites that regulate transcription and chromatin organization. Despite evidence linking CTCF to the protection of epigenetic states through barrier insulation, the impact of CTCF loss on genome-wide DNA methylation sites in human cancer remains undefined. Results: Here, we demonstrate that prostate and breast cancers within The Cancer Genome Atlas (TCGA) exhibit frequent copy number loss of CTCF and that this loss is associated with increased DNA methylation events that occur preferentially at CTCF binding sites. CTCF sites differ among tumor types and result in tissue-specific methylation patterns with little overlap between breast and prostate cancers. DNA methylation and transcriptome profiling in vitro establish that forced downregulation of CTCF leads to spatially distinct DNA hypermethylation surrounding CTCF binding sites, loss of CTCF binding, and decreased gene expression that is also seen in human tumors. DNA methylation inhibition reverses loss of expression at these CTCF-regulated genes. Conclusion: These findings establish CTCF loss as a major mediator in directing localized DNA hypermethylation events in a tissue-specific fashion and further support its role as a driver of the cancer phenotype.

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Section: Methylation Analyses Including Combined Bisulfite Restrictiomentioning

confidence: 99%

CTCF loss mediates unique DNA hypermethylation landscapes in human cancers

et al. 2020

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“…Figure B shows the quantification of the actual allele expression by PIE from mouse tail cDNA precisely reflects theoretical expression, as the R‐squared value for the trendline is 0.996. The sensitivities of PIE using mouse and human gDNA showed higher R 2 values than those generated from cDNA [Yang et al, ]. In both assays, PIE was able to detect 1% differences in allelic ratios.…”

Section: Resultsmentioning

confidence: 99%

“…We utilized a previously identified A/G polymorphism in IGF2 located on exon 5 in the human transcript and exon 6 in the mouse. Approaches have evaluated allelic expression on these loci with both Apa I digestion [Fu et al, ], Flu‐PE [Fu et al, ], and pyrosequencing [Yang et al, ]. Given the previous data on homozygous and heterozygous individuals for both loci, the sensitivity and specificity of the assays were able to be compared.…”

Section: Methodsmentioning

confidence: 99%

Pyrosequencing for Accurate Imprinted Allele Expression Analysis

Yang

Damaschke

Yao

et al. 2015

J of Cellular Biochemistry

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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.

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DNA Methylation Analysis by Pyrosequencing

Busato¹

2014

Encyclopedia of Metagenomics

447

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Pyrosequencing for the rapid and efficient quantification of allele-specific expression

Cited by 7 publications

References 18 publications

CTCF loss mediates unique DNA hypermethylation landscapes in human cancers

CTCF loss mediates unique DNA hypermethylation landscapes in human cancers

Pyrosequencing for Accurate Imprinted Allele Expression Analysis

DNA Methylation Analysis by Pyrosequencing

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