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Quantification of Unmethylated Insulin DNA Using Methylation Sensitive Restriction Enzyme Digital Polymerase Chain Reaction

Assessment of specific β-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 β-cell...

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Autores principales: van de Leemkolk, Fenna E. M., Nell, Rogier J., Versluis, Mieke, de Koning, Eelco J. P., Huurman, Volkert A. L., Alwayn, Ian P. J., Ploeg, Rutger J., van der Velden, Pieter A., Engelse, Marten A.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9022224/
https://www.ncbi.nlm.nih.gov/pubmed/35462792
http://dx.doi.org/10.3389/ti.2022.10167
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author van de Leemkolk, Fenna E. M.
Nell, Rogier J.
Versluis, Mieke
de Koning, Eelco J. P.
Huurman, Volkert A. L.
Alwayn, Ian P. J.
Ploeg, Rutger J.
van der Velden, Pieter A.
Engelse, Marten A.
author_facet van de Leemkolk, Fenna E. M.
Nell, Rogier J.
Versluis, Mieke
de Koning, Eelco J. P.
Huurman, Volkert A. L.
Alwayn, Ian P. J.
Ploeg, Rutger J.
van der Velden, Pieter A.
Engelse, Marten A.
author_sort van de Leemkolk, Fenna E. M.
collection PubMed
description Assessment of specific β-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 β-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 β-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 β-cells.
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spelling pubmed-90222242022-04-22 Quantification of Unmethylated Insulin DNA Using Methylation Sensitive Restriction Enzyme Digital Polymerase Chain Reaction van de Leemkolk, Fenna E. M. Nell, Rogier J. Versluis, Mieke de Koning, Eelco J. P. Huurman, Volkert A. L. Alwayn, Ian P. J. Ploeg, Rutger J. van der Velden, Pieter A. Engelse, Marten A. Transpl Int Health Archive Assessment of specific β-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 β-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 β-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 β-cells. Frontiers Media S.A. 2022-04-07 /pmc/articles/PMC9022224/ /pubmed/35462792 http://dx.doi.org/10.3389/ti.2022.10167 Text en Copyright © 2022 van de Leemkolk, Nell, Versluis, de Koning, Huurman, Alwayn, Ploeg, van der Velden and Engelse. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Health Archive
van de Leemkolk, Fenna E. M.
Nell, Rogier J.
Versluis, Mieke
de Koning, Eelco J. P.
Huurman, Volkert A. L.
Alwayn, Ian P. J.
Ploeg, Rutger J.
van der Velden, Pieter A.
Engelse, Marten A.
Quantification of Unmethylated Insulin DNA Using Methylation Sensitive Restriction Enzyme Digital Polymerase Chain Reaction
title Quantification of Unmethylated Insulin DNA Using Methylation Sensitive Restriction Enzyme Digital Polymerase Chain Reaction
title_full Quantification of Unmethylated Insulin DNA Using Methylation Sensitive Restriction Enzyme Digital Polymerase Chain Reaction
title_fullStr Quantification of Unmethylated Insulin DNA Using Methylation Sensitive Restriction Enzyme Digital Polymerase Chain Reaction
title_full_unstemmed Quantification of Unmethylated Insulin DNA Using Methylation Sensitive Restriction Enzyme Digital Polymerase Chain Reaction
title_short Quantification of Unmethylated Insulin DNA Using Methylation Sensitive Restriction Enzyme Digital Polymerase Chain Reaction
title_sort quantification of unmethylated insulin dna using methylation sensitive restriction enzyme digital polymerase chain reaction
topic Health Archive
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9022224/
https://www.ncbi.nlm.nih.gov/pubmed/35462792
http://dx.doi.org/10.3389/ti.2022.10167
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