Measurement of Differentially Methylated INS DNA Species in Human Serum Samples as a Biomarker of Islet β Cell Death

dc.contributor.authorTersey, Sarah A.
dc.contributor.authorNelson, Jennifer B.
dc.contributor.authorFisher, Marisa M.
dc.contributor.authorMirmira, Raghavendra G.
dc.contributor.departmentDepartment of Pediatrics, IU School of Medicineen_US
dc.date.accessioned2017-08-02T16:56:24Z
dc.date.available2017-08-02T16:56:24Z
dc.date.issued2016-12-21
dc.description.abstractThe death of islet β cells is thought to underlie the pathogenesis of virtually all forms of diabetes and to precede the development of frank hyperglycemia, especially in type 1 diabetes. The development of sensitive and reliable biomarkers of β cell death may allow for early therapeutic intervention to prevent or delay the development of diabetes. Recently, several groups including our own have reported that cell-free, differentially methylated DNA encoding preproinsulin (INS) in the circulation is correlated to β cell death in pre-type 1 diabetes and new-onset type 1 diabetes. Here, we present a step-by-step protocol using digital PCR for the measurement of cell-free INS DNA that is differentially methylated at cytosine at position -69 bp (relative to the transcriptional start site). We demonstrate that the assay can distinguish between methylated and unmethylated cytosine at position -69 bp, is linear across several orders of magnitude, provides absolute quantitation of DNA copy numbers, and can be applied to samples of human serum from individuals with new-onset type 1 diabetes and disease-free controls. The protocol described here can be adapted to any DNA species for which detection of differentially methylated cytosines is desired, whether from circulation or from isolated cells and tissues, and can provide absolute quantitation of DNA fragments.en_US
dc.eprint.versionAuthor's manuscripten_US
dc.identifier.citationTersey, S. A., Nelson, J. B., Fisher, M. M., & Mirmira, R. G. (2016). Measurement of Differentially Methylated INS DNA Species in Human Serum Samples as a Biomarker of Islet β Cell Death. Journal of Visualized Experiments : JoVE, (118), 10.3791/54838. Advance online publication. http://doi.org/10.3791/54838en_US
dc.identifier.urihttps://hdl.handle.net/1805/13720
dc.language.isoen_USen_US
dc.publisherJoVEen_US
dc.relation.isversionof10.3791/54838en_US
dc.relation.journalJournal of Visualized Experiments : JoVEen_US
dc.rightsPublisher Policyen_US
dc.sourcePMCen_US
dc.subjectGeneticsen_US
dc.subjectIssue 118en_US
dc.subjectBiomarkeren_US
dc.subjectIsleten_US
dc.subjectDiabetesen_US
dc.subjectInsulinen_US
dc.subjectDigital PCRen_US
dc.subjectEpigeneticsen_US
dc.titleMeasurement of Differentially Methylated INS DNA Species in Human Serum Samples as a Biomarker of Islet β Cell Deathen_US
dc.typeArticleen_US
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