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... 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
Nell, R.J.; Steenderen, D. van; Menger, N.V.; Weitering, T.J.; Versluis, M.; Velden, P.A. van der 2020
Epigenetic regulation is important in human health and disease, but the exact mechanisms remain largely enigmatic. DNA methylation represents one epigenetic aspect but is challenging to quantify.... Show moreEpigenetic regulation is important in human health and disease, but the exact mechanisms remain largely enigmatic. DNA methylation represents one epigenetic aspect but is challenging to quantify. In this study, we introduce a digital approach for the quantification of the amount and density of DNA methylation. We designed an experimental setup combining efficient methylation-sensitive restriction enzymes with digital polymerase chain reaction (PCR) to quantify a targeted density of DNA methylation independent of bisulfite conversion. By using a stable reference and comparing experiments treated and untreated with these enzymes, copy number instability could be properly normalized. In silico simulations demonstrated the mathematical validity of the setup and showed that the measurement precision depends on the amount of input DNA and the fraction methylated alleles. This uncertainty could be successfully estimated by the confidence intervals. Quantification ofRASSF1promoter methylation in a variety of healthy and malignant samples and in a calibration curve confirmed the high accuracy of our approach, even in minute amounts of DNA. Overall, our results indicate the possibility of quantifying DNA methylation with digital PCR, independent of bisulfite conversion. Moreover, as the context-density of methylation can also be determined, biological mechanisms can now be quantitatively assessed. Show less
Colombo, M.; Lopez-Perolio, I.; Meeks, H.D.; Caleca, L.; Parsons, M.T.; Li, H.Y.; ... ; Study HEBON 2018
