Validation of methylation-sensitive high resolution melting for the detection of DNA methylation in cholangiocarcinoma

Amornpisutt, Rattaya; Sriraksa, Ruethairat; Limpaiboon, Temduang

doi:10.1016/j.clinbiochem.2012.04.027

Cited by 15 publications

(17 citation statements)

References 10 publications

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“…Although MS-HRM is unable to detect methylation level at individual CpG sites as determined by pyrosequencing, our previous study has shown that methylation levels obtained from MS-HRM were positively correlated with those from pyrosequencing [29]. Accordingly, MS-HRM enables to identify methylation status of homogenous, heterogeneous, or partial methylation which is recognizable by its characteristic melting profiles.…”

Section: Discussionmentioning

confidence: 88%

“…Methylation levels of OPCML and SFRP1 in CCA samples and adjacent normal tissues With regard to our previous study, MS-HRM is a new and acceptable approach with high reproducibility to apply for quantification of DNA methylation in CCA [29]. The methylation levels of OPCML and SFRP1 in 73 CCA and 10 matched adjacent normal tissues quantified by MS-HRM are illustrated in Fig.…”

Section: Quantification Of Dna Methylation By Ms-hrmmentioning

confidence: 97%

“…For OPCML (GenBank ID: NM_002545, target's location: Chromosome 11: 132,944, 392-132,944,526), the primer sequences for MS-HRM were obtained from our previous study [29].…”

Section: Methylation-sensitive High-resolution Melting (Ms-hrm)mentioning

confidence: 99%

“…Methylation-sensitive high-resolution melting (MS-HRM) is a novel close tube assay for assessment of methylation level which has been recently established [28] and popularly applied in epigenetic cancer research over the past few years. Our previous study has validated MS-HRM and shown that it is comparable to pyrosequencing in terms of quantification of DNA methylation in clinical samples [29]. Regarding its simple, robust, and cost-effective technique, we have applied sophisticated MS-HRM to quantify the methylation level of two candidate loci OPCML and SFRP1 in CCA patients in comparison with adjacent normal tissues, and further evaluated the sensitivity, specificity, and accuracy of the assays.…”

Section: Introductionmentioning

confidence: 98%

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DNA methylation level of OPCML and SFRP1: a potential diagnostic biomarker of cholangiocarcinoma

et al. 2015

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Cholangiocarcinoma (CCA) is a malignancy of the bile duct epithelium which is caused by liver fluke infection. The clinical symptoms of CCA were revealed as the disease progresses to advanced stage. Thus, specific diagnostic biomarkers are important for this fatal disease. We applied methylation-sensitive high-resolution melting (MS-HRM) to quantify DNA methylation levels of opioid binding protein/cell adhesion molecule-like gene (OPCML) and Secreted frizzled-related protein 1 (SFRP1) in 73 primary CCA and 10 adjacent normal tissues and evaluated the sensitivity, specificity, and accuracy of the assay. The median methylation level of OPCML in CCA was 38.7 % (ranged from 0 to 82.2 %) and of SFRP1 was 31.5 % (ranged from 0 to 86.2 %). Methylation cutoff values of OPCML and SFRP1 derived from adjacent normal tissue were 6.90 and 10.44 %, respectively. With these cutoff values, the area under curve (AUC) of OPCML was 0.932 (95 % CI 0.878-0.986) and of SFRP1 was 0.951 (95 % CI 0.905-0.996). The sensitivity, specificity, and accuracy of OPCML were 89.04, 100, and 90.36 %, respectively, and of SFRP1 were 83.56, 100, and 85.54 %, respectively. In conclusion, the DNA methylation levels of OPCML and SFRP1 could be potential biomarkers for diagnosis of CCA with high specificity, sensitivity, and accuracy, in particular for biopsy specimens. Further validation in noninvasive samples such as serum or plasma is warranted for clinical applicability, especially as early diagnostic biomarkers.

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Section: Discussionmentioning

confidence: 88%

Section: Quantification Of Dna Methylation By Ms-hrmmentioning

confidence: 97%

“…For OPCML (GenBank ID: NM_002545, target's location: Chromosome 11: 132,944, 392-132,944,526), the primer sequences for MS-HRM were obtained from our previous study [29].…”

Section: Methylation-sensitive High-resolution Melting (Ms-hrm)mentioning

confidence: 99%

Section: Introductionmentioning

confidence: 98%

See 2 more Smart Citations

DNA methylation level of OPCML and SFRP1: a potential diagnostic biomarker of cholangiocarcinoma

et al. 2015

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“…Since the energy needed to break the hydrogen bonds between the hybridized DNA strands depends on the sequence characteristics such as length and guaninecytosine content (GC), the melting profile is intimately correlated with the amplicon sequence. Recently, HRM has also been extended to applications in DNA methylation detection and quantification [6][7][8][9][10][11][12][13][14] by combining it with bisulfite conversion and PCR. Bisulfite conversion is a technique commonly employed in the study of DNA methylation, where unmethylated cytosines in DNA samples are chemically converted into uracils, while methylated cytosines are preserved (Fig.…”

Section: Introductionmentioning

confidence: 99%