Quantification of unmethylated insulin DNA using methylation sensitive restriction enzyme digital polymerase chain reaction

Leemkolk, F.E.M. van de; Nell, R.J.; Versluis, M.; Koning, E.J.P. de; Huurman, V.A.L.; Alwayn, I.P.J.; Ploeg, R.J.; Velden, P.A. van der; Engelse, M.A.

doi:doi:10.3389/ti.2022.10167

Assessment of specific beta-cell death can be used to determine the quality and viability of pancreatic islets prior to transplantation and hence predict the suitability of the pancreas for isolation. Recently, several groups have demonstrated that unmethylated insulin (INS)-DNA is correlated to beta-cell death in type 1 diabetes patients and during clinical islet isolation and subsequent transplantation. Here, we present a step-by-step protocol of our novel developed method for quantification of the relative amount of unmethylated INS-DNA using methylation sensitive restriction enzyme digital polymerase chain reaction This method provides a novel and sensitive way to quantify the relative amount of beta-cell derived unmethylated INS-DNA in cellular lysate. We therefore suggest that this technique can be of value to reliably determine the purity of an islet preparation and may also serve as a measure of the quality of islets prior to transplantation measuring unmethylated INS-DNA as...Show moreAssessment of specific beta-cell death can be used to determine the quality and viability of pancreatic islets prior to transplantation and hence predict the suitability of the pancreas for isolation. Recently, several groups have demonstrated that unmethylated insulin (INS)-DNA is correlated to beta-cell death in type 1 diabetes patients and during clinical islet isolation and subsequent transplantation. Here, we present a step-by-step protocol of our novel developed method for quantification of the relative amount of unmethylated INS-DNA using methylation sensitive restriction enzyme digital polymerase chain reaction This method provides a novel and sensitive way to quantify the relative amount of beta-cell derived unmethylated INS-DNA in cellular lysate. We therefore suggest that this technique can be of value to reliably determine the purity of an islet preparation and may also serve as a measure of the quality of islets prior to transplantation measuring unmethylated INS-DNA as a reflection of the relative amount of lysed beta-cells.Show less

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Quantification of unmethylated insulin DNA using methylation sensitive restriction enzyme digital polymerase chain reaction