Detection of methylation in promoter sequences by melting curve analysis-based semiquantitative real time PCR

Lorente, Aiala; Mueller, Wolf; Urdangarín, Edurne; Lázcoz, Paula; Deimling, Andreas von; Castresana, Javier S.

doi:10.1186/1471-2407-8-61

Cited by 55 publications

(40 citation statements)

References 19 publications

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“…Genomic DNA quantification was performed on a NanoVue Spectrophotometer (GE LifeSciences, Piscataway, NJ, USA). The basic principle of bisulfite modification of DNA is that all unmethylated cytosines are deaminated and sulfonated, converting them to thymines, whereas methylated cytosines (5-methyl-cytosines) remain unaltered in the bisulfite reaction (Lorente et al, 2008).…”

Section: Deoxyribonucleic Acid Extraction and Bisulfite Modificationmentioning

confidence: 99%

Epigenetic DNA methylation in the promoters of Peroxisome Proliferator-Activated Receptor γ in chicken lines divergently selected for fatness1

Sun

Gao

Qiao

et al. 2014

Journal of Animal Science

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Peroxisome proliferator-activated receptor γ is a master regulator of adipocyte differentiation and function. Expression of PPARγ in mammals is regulated by DNA methylation; however, it is currently unknown whether chicken PPARγ expression is regulated by DNA methylation. To enhance our understanding of molecular mechanisms underlying chicken adipose tissue development and adipogenesis, we investigated the promoter methylation status and gene expression of PPARγ gene in Northeast Agricultural University broiler lines divergently selected for abdominal fat content (NEAUHLF). Deoxyribonucleic acid methylation was analyzed by bisulfite sequencing method, and mRNA expression was detected by real-time quantitative real time reverse-transcription polymerase chain reaction (RT-PCR). The analyzed region located from -1,175 to -301 bp upstream of the translation start codon ATG contains 6 CpG dinucleotides, which are located at positions -1,014, -796, -625, -548, -435, and -383 bp, respectively. The results revealed that the 3 CpGs at positions -548, -435, and -383 bp showed differential methylation between the lean and fat chicken lines, but the other 3 CpG sites at positions -1,014, -796, and -625 bp did not. PPARγ gene promoter methylation in both chicken lines decreased with age, and PPARγ promoter methylation levels were significantly higher in lean than fat broilers at 2 wk of age (79.9 to 64.5%; P < 0.0001), at 3 wk of age (66.7 to 58.3%; P < 0.0001), and at 7 wk of age (50.0 to 42.7%; P = 0.0004). Real-time quantitative RT-PCR analysis showed that, negatively correlated with DNA methylation (Pearson's r = -0.653, P = 0.0057), PPARγ expression was increased with age and significantly lower in lean than fat chicken lines at 2, 3, and 7 wk of age (P < 0.0001). In conclusion, our findings suggest that chicken PPARγ is regulated by DNA methylation during adipose tissue development.

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Section: Deoxyribonucleic Acid Extraction and Bisulfite Modificationmentioning

confidence: 99%

Epigenetic DNA methylation in the promoters of Peroxisome Proliferator-Activated Receptor γ in chicken lines divergently selected for fatness1

Sun

Gao

Qiao

et al. 2014

Journal of Animal Science

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“…To determine the promoter methylation status of CDKN2A/p16, melting curve analysis-based semi-quantitative real time PCR was performed as previously described (Lorente et al, 2008;Smith et al, 2009). In short: genomic DNA from all samples was modified with bisulfate to convert only unmethylated cytosine residues into uracil residues.…”

Section: Melting Curve Analysis-methylation Assay (Mca-meth)mentioning

confidence: 99%

Small deletions but not methylation underlie CDKN2A/p16 loss of expression in conventional osteosarcoma

Mohseny

Tieken

Velden

et al. 2010

Genes Chromosomes & Cancer

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Conventional osteosarcoma is characterized by rapid growth, high local aggressiveness, and metastasizing potential. Patients developing lung metastases experience poor prognosis despite extensive chemotherapy regimens and surgical interventions. Previously we identified a subgroup of osteosarcoma patients with loss of CDKN2A/p16 protein expression in the primary tumor biopsies which was significantly predictive of a very poor prognosis. Here we aimed to identify the underlying mechanism(s) of this protein loss in relation to osteosarcoma behavior. The CDKN2A locus was analyzed in osteosarcoma cases with total loss of CDKN2A/p16 expression and in cases with high protein expression using melting curve analysis-methylation assay (MCA-Meth), fluorescent in situ hybridization (FISH), multiplex ligation-dependent probe amplification (MLPA), and mutation analysis. All cases with complete CDKN2A/p16 protein loss showed homozygous deletions at the CDKN2A locus. In none of the cases hyper methylation of the promoter region was seen which was confirmed by sequencing this region. Taken together we show that large or smaller deletions of the CDKN2A locus are evident in patient samples and underlie the CDKN2A/p16 protein expression loss while promoter methylation does not appear to be a mechanism of this expression loss. Genomic loss of CDKN2A instead of promoter methylation might be a plausible explanation for the rapid proliferation and high aggressiveness of osteosarcoma by simultaneous impairment CDKN2A/p14(ARF) function.

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“…Bisulfite sequencing (BS) and MSP were done as previously described (13)(14)(15) and used as additional independent techniques for validation and investigation of the DR4 promoter region. MSP primers were designed based on DR4 promoter sequencing data and on previously published data on DR4 promoter methylation (16).…”

Section: Methodsmentioning

confidence: 99%

Epigenetic Silencing of Death Receptor 4 Mediates Tumor Necrosis Factor–Related Apoptosis-Inducing Ligand Resistance in Gliomas

Elias

Siegelin

Steinmüller

et al. 2009

Clinical Cancer Research

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Purpose: To identify and characterize epigenetically regulated genes able to predict sensitivity or resistance to currently tested chemotherapeutic agents in glioma therapy. Experimental Design: We used methylation-sensitive BeadArray technology to identify novel epigenetically regulated genes associated with apoptosis and with potential therapeutic targets in glioma therapy. To elucidate the functional consequences of promoter methylation in the identified target death receptor 4 (DR4), we investigated tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated and anti-DR4-mediated apoptosis in glioma cell lines (U373 and A172) with loss of DR4 and one glioma cell line (LN18) with robust DR4 expression. Results: In human astrocytic tumors, we detected DR4 promoter hypermethylation in 60% (n = 5) of diffuse astrocytomas WHO grade 2, in 75% (n = 8) of anaplastic astrocytomas WHO grade 3, and in 70% of glioblastomas WHO grade 4 (n = 33). DR4 is a cell surface protein restricted to glioma cells and is targeted by TRAIL. Glioma cell lines U373 and A172 harbored heavily methylated DR4 promoters, and 5-aza-2-deoxycytidinemediated demethylation reconstituted DR4 expression in these cell lines. Functional knockdown of DR4 by DR4-specific small interfering RNA in TRAIL-sensitive glioma cell line LN18 significantly mitigated apoptosis induced by an agonistic anti-DR4 antibody. 5-Aza-2-deoxycytidine-mediated demethylation resulted in a functional reconstitution of DR4 on the cell surface of TRAIL-resistant glioma cell line U373 and sensitized U373 to TRAIL-mediated apoptosis. Suppression of DR4 by small interfering RNA in demethylated U373 successfully reestablished the TRAIL-resistant phenotype of U373. Conclusions: DR4 promoter methylation is frequent in human astrocytic gliomas, and epigenetic silencing of DR4 mediates resistance to TRAIL/DR4-based glioma therapies. Epigenetic modifications contribute to tumorigenesis. Examples are epigenetically silenced tumor suppressor genes (1). However, the epigenome becomes crucial clinical relevance when epigenetically regulated proteins are identified that themselves constitute therapeutic targets or are responsible for the development of therapy resistance of the tumor. An example for the latter is O 6 -methylguanine DNA methyltransferase (MGMT) in glioma therapy. Epigenetic silencing of MGMT abolishes its capability of chemotherapy resistance and predicts therapeutic response to alkylating drugs in glioma therapy (2-4). Caspase-8 may exemplify a therapeutically targeted epigenetically silenced molecule in gliomas. A downstream target for tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis, loss of caspase-8 expression, has been associated with epigenetic silencing and TRAIL resistance in these tumors (5). We were interested in identifying additional epigenetically silenced proteins or receptor molecules that are already targeted in glioma therapy. To address this, we implemented single-nucleotide polymorphism-based BeadArra...

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Detection of methylation in promoter sequences by melting curve analysis-based semiquantitative real time PCR

Cited by 55 publications

References 19 publications

Epigenetic DNA methylation in the promoters of Peroxisome Proliferator-Activated Receptor γ in chicken lines divergently selected for fatness1

Epigenetic DNA methylation in the promoters of Peroxisome Proliferator-Activated Receptor γ in chicken lines divergently selected for fatness1

Small deletions but not methylation underlie CDKN2A/p16 loss of expression in conventional osteosarcoma

Epigenetic Silencing of Death Receptor 4 Mediates Tumor Necrosis Factor–Related Apoptosis-Inducing Ligand Resistance in Gliomas

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