Dataset Information

A closed-tube methylation-sensitive high resolution melting assay (MS-HRMA) for the semi-quantitative determination of CST6 promoter methylation in clinical samples.

ABSTRACT:

Background

CST6 promoter is highly methylated in cancer, and its detection can provide important prognostic information in breast cancer patients. The aim of our study was to develop a Methylation-Sensitive High Resolution Melting Analysis (MS-HRMA) assay for the investigation of CST6 promoter methylation.

Methods

We designed primers that amplify both methylated and unmethylated CST6 sequences after sodium bisulfate (SB) treatment and used spiked control samples of fully methylated to unmethylated SB converted genomic DNA to optimize the assay. We first evaluated the assay by analyzing 36 samples (pilot training group) and further analyzed 80 FFPES from operable breast cancer patients (independent group). MS-HRMA assay results for all 116 samples were compared with Methylation-Specific PCR (MSP) and the results were comparable.

Results

The developed assay is highly specific and sensitive since it can detect the presence of 1% methylated CST6 sequence and provides additionally a semi-quantitative estimation of CST6 promoter methylation. CST6 promoter was methylated in 39/80 (48.75%) of FFPEs with methylation levels being very different among samples. MS-HRMA and MSP gave comparable results when all samples were analyzed by both assays.

Conclusions

The developed MS-HRMA assay for CST6 promoter methylation is closed tube, highly sensitive, cost-effective, rapid and easy-to-perform. It gives comparable results to MSP in less time, while it offers the advantage of additionally providing an estimation of the level of methylation.

SUBMITTER: Dimitrakopoulos L

PROVIDER: S-EPMC3495665 | biostudies-literature | 2012 Oct

REPOSITORIES: biostudies-literature

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Publications

A closed-tube methylation-sensitive high resolution melting assay (MS-HRMA) for the semi-quantitative determination of CST6 promoter methylation in clinical samples.

Dimitrakopoulos Lampros L Vorkas Panagiotis A PA Georgoulias Vasilis V Lianidou Evi S ES

BMC cancer 20121022

<h4>Background</h4>CST6 promoter is highly methylated in cancer, and its detection can provide important prognostic information in breast cancer patients. The aim of our study was to develop a Methylation-Sensitive High Resolution Melting Analysis (MS-HRMA) assay for the investigation of CST6 promoter methylation.<h4>Methods</h4>We designed primers that amplify both methylated and unmethylated CST6 sequences after sodium bisulfate (SB) treatment and used spiked control samples of fully methylate ...[more]

PMID: 23088560

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Project description:The emergence of novel drugs and the continuous expansion of the scope of the types of drugs under control have greatly increased requests for screening of a range of drugs in hair. Here, a multi-analyte method for the detection and quantification of 88 psychotropic drugs in the hair of addicts in drug abstinence was developed and fully validated using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Hair samples (25 mg) were washed, cut into pieces, cryogenically ground, and extracted in methanol. The extracted analytes were separated on an Allure PFPP column (100×2.1 mm, 5 mm i.d., Restek, USA) and analyzed by LC-MS/MS in multiple reaction monitoring mode. The limits of detection and limits of quantification ranged from 0.1 to 20 pg/mg and 0.2 to 50 pg/mg, respectively. The intra- and inter-assay precisions (RSD) of all analysis ranged from 0.9 to 14.9% and 1.9 to 15.9%, respectively. Accuracy values were 100±20%. The extraction recovery of quality control samples ranged from 50.9% to 99.6% for all analytes. The matrix effects for all analytes ranged from 46.8% to 99.7%. The method was successfully used to analyze 1,865 hair samples from addicts in drug rehabilitation at their own communities.. Among the samples, 129 cases were positive; the majority of positive cases were from males (78.29%), 92.25% of whom were over 35 years old. Traditional drugs, like methamphetamine and opioids, accounted for most positive cases, and 27 of the abstinence cases with a use history of methamphetamine were still positive. In addition to abused drugs, like methamphetamine, morphine, and cocaine, the sedative hypnotic and psychotherapeutic drugs, including clonazepam, alprazolam, estazolam, zolpidem, and quetiapine, were detected in 26% of the hair samples, suggesting that these addicts may have insomnia and mental problems such as depression and psychosis, probably due to the long-term effects of drugs and withdrawal reactions. Three synthetic cannabinoids were also detected in 4 (2.7%) cases. A total of 37 cases were positive for methadone, tramadol, and dextromethorphan, reflecting a new trend of alternative drug use when traditional drugs were not easy to obtain during the COVID-19 outbreak.

Dataset Information

A closed-tube methylation-sensitive high resolution melting assay (MS-HRMA) for the semi-quantitative determination of CST6 promoter methylation in clinical samples.

Background

Methods

Results

Conclusions

Publications

A closed-tube methylation-sensitive high resolution melting assay (MS-HRMA) for the semi-quantitative determination of CST6 promoter methylation in clinical samples.

Similar Datasets

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