A quantitative method for detecting DNA methylation over targeted genomic regions using isotope dilution liquid chromatography tandem mass spectrometry

Ye, Xiaoxia; Zhang, Lijian; Chen, Bin; Li, Jian; Yang, Qingjin; Huang, Qionglin; Zhang, Junjie; Gao, Yongping; Li, Zhaojin; Cai, Chun

doi:10.1016/j.talanta.2017.03.082

Cited by 12 publications

(15 citation statements)

References 21 publications

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“…To ensure the accuracy of the LC‐MS/MS method, 3 methylation standards including 0%, 47%, and 100% methylation levels were prepared according to our prior work . With the primers and cycling conditions listed in Table S2, the exon 2 region of CDKN2A gene was amplified by using template DNA extracted from tumor‐adjacent tissue, human intestinal epithelial cell line FHs74Int, and human colon cancer cell line HCT116, respectively.…”

Section: Methodsmentioning

confidence: 99%

“…An LC‐MS/MS method with excellent accuracy, which was described in our former publication, was applied to determine the methylation levels of aforementioned target regions.…”

Section: Methodsmentioning

confidence: 99%

“…The levels of regional DNA methylation were calculated based on the LC‐MS/MS data by using the formula shown below.

normal% Methylation = ((), P_{GuaCyt - normalM} - P_{Gua - normalD}) true/ P_{Cyt - CpG - normalD} \times 100 % and P_{GuaCyt - normalM} = Q_{Cyt - normalM} true/ ((), Q_{Cyt - normalM} + Q_{Ade - normalM})

where P GuaCyt‐M is the molar percent of combined guanine and Cyt in bisulfite PCR amplicon, Q Cyt‐M and Q Ade‐M are the molar quantity of Cyt and Ade determined in DNA samples, P Gua‐D is the molar percent of guanine to total bases in original sequences of targeted region, and P Cyt‐CpG‐D is the molar percent of Cyt in CpG sites to total bases in original sequences of the targeted region.…”

Section: Methodsmentioning

confidence: 99%

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Screening of exon methylation biomarkers for colorectal cancer via LC‐MS/MS strategy

Huang

Yang

et al. 2017

J Mass Spectrom

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The identification of biomarkers would be of benefit for the diagnosis and treatment of colorectal cancer. DNA methylation in specific genomic regions, which had shown strongly association with disease genotypes, was an effective indicator to reveal the occurrence and development of cancers. To screen out methylation biomarkers for colorectal cancer (CRC), genomic DNA was isolated from colorectal cancerous and corresponding cancer-adjacent tissues collected from 30 CRC patients and then bisulfite-converted. The exon regions of 5 targeted genes (CNRIP1, HIC1, RUNX3, p15, and SFRP2) were amplified by using nested polymerase chain reaction with specific primers, and the amplicon was purified and hydrolyzed. The methylation levels of these specific regions were detected by liquid chromatography tandem mass spectrometry (LC-MS/MS). The results showed that 5 targeted exon regions were successfully amplified and confirmed by sequencing. The methodological validations indicated that LC-MS/MS was highly sensitive and accurate. The methylation levels of CNRIP1 and RUNX3 were remarkably high in CRC tissues with statistical difference when compared with corresponding cancer-adjacent individuals, while that of HIC1, p15, and SFRP2 had no difference between 2 subjects. These findings supported CNRIP1 and RUNX3 as potential DNA methylation biomarkers for CRC diagnosis and treatment, and our LC-MS/MS approach exhibited great advantages in the identification of regional DNA methylation biomarkers.

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Section: Methodsmentioning

confidence: 99%

“…An LC‐MS/MS method with excellent accuracy, which was described in our former publication, was applied to determine the methylation levels of aforementioned target regions.…”

Section: Methodsmentioning

confidence: 99%

“…The levels of regional DNA methylation were calculated based on the LC‐MS/MS data by using the formula shown below.

normal% Methylation = ((), P_{GuaCyt - normalM} - P_{Gua - normalD}) true/ P_{Cyt - CpG - normalD} \times 100 % and P_{GuaCyt - normalM} = Q_{Cyt - normalM} true/ ((), Q_{Cyt - normalM} + Q_{Ade - normalM})

Section: Methodsmentioning

confidence: 99%

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Screening of exon methylation biomarkers for colorectal cancer via LC‐MS/MS strategy

Huang

Yang

et al. 2017

J Mass Spectrom

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“…According to Scopus, the number of publications on LC-MS had dramatically increased since 2006. LC-MS has a variety of applications and can be extended to wide a range of target molecules such as metabolites [1][2][3][4][5][6][7] and biological polymers [7][8][9][10][11][12][13]. This technique was also used to identify unknown compounds in natural products, e.g.…”

Section: Applications Of Lc-msmentioning

confidence: 99%

Hydrophilic Interaction Liquid Chromatography for LC-MS

Hanai¹

2018

Mass Spectrom Purif Tech

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The retention mechanisms in hydrophilic interaction liquid chromatography (HILIC) are quantitatively described in in silico analysis. Among a variety of polar bonded-phase silica gels, hexylamine-bonded silica gel is stable and can be used in both aqueous HILIC and ion-exchange liquid chromatography with high reproducibility. Hexynyl and phenylhexyl bonded silica gels are also used in HILIC with high reproducibility. The retention mechanisms in aqueous hydrophilic and non-aqueous hydrophilic (normal-phase) liquid chromatography are similar. Both hydrogenbonding and weak electrostatic interaction contributes to retention. Furthermore, strong electrostatic interaction is dominant in ion-exchange liquid chromatography. A suitable liquid chromatographic system for liquid chromatography (LC)-mass spectrophotometer (MS) is proposed.

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“…To explore the methylation changes of p16 gene body in CRC, we focused on CpG-rich regions in p16 gene body, namely exon 1 and exon 2. Their methylation levels were evaluated in paired CRC and adjacent normal tissues by LC-MS/MS method, which can quantify the average methylation level of target genomic region [ 30 ]. Statistical analysis was carried out to find more reliable methylation biomarkers.…”

Section: Introductionmentioning

confidence: 99%

The hypermethylation of p16 gene exon 1 and exon 2: potential biomarkers for colorectal cancer and are associated with cancer pathological staging

et al. 2018

BMC Cancer

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BackgroundTumor suppressor gene p16 promoter hypermethylation has been widely studied in colorectal cancer (CRC), yet its clinicopathological significance remains controversial. The methylation alterations of other regions within p16 gene are still rarely researched. The present study aimed to explore the methylation changes of p16 gene body in CRC and to find whether they were associated with clinicopathological staging of CRC.MethodsPaired colorectal cancer tissues and corresponding adjacent normal tissues from 30 CRC patients were collected. The methylation levels of two CpG islands within p16 gene body, exon 1 and exon 2, were accurately assessed simultaneously by a LC-MS/MS method. The p16 protein expressions were assessed by immunohistochemistry assay. Statistical analyses were carried out using SPSS 17.0 software. Heat-map analysis was carried out by HemI 1.0 software.ResultsIn the present study, CRC tissues showed more highly methylated than adjacent normal tissues at both CpG islands of p16 gene. And exon 2 hypermethylation was higher and more frequent than exon 1. The ROC curve analysis showed that the simultaneous use of both indicators had excellent sensitivity and specificity for distinguishing CRC tissues and adjacent normal tissues. Following, the methylation level of p16 exon 1/2 was negatively related to p16 protein expression. Further correlation analysis revealed that p16 exon 1 hypermethylation was associated with N/Dukes staging (p = 0.033), and p16 exon 2 hypermethylaiton was associated with T staging (p = 0.035).ConclusionsThe p16 gene body was remarkably hyper-methylated in CRC tissues and associated with p16 protein expression and cancer clinicopathological staging. The combination of p16 exon 1 and exon 2 could better reflect the overall methylation status of p16 gene body and provide potential biomarkers of CRC.

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A quantitative method for detecting DNA methylation over targeted genomic regions using isotope dilution liquid chromatography tandem mass spectrometry

Cited by 12 publications

References 21 publications

Screening of exon methylation biomarkers for colorectal cancer via LC‐MS/MS strategy

Screening of exon methylation biomarkers for colorectal cancer via LC‐MS/MS strategy

Hydrophilic Interaction Liquid Chromatography for LC-MS

The hypermethylation of p16 gene exon 1 and exon 2: potential biomarkers for colorectal cancer and are associated with cancer pathological staging

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