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

Abstract

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.

Keywords: biomarker; digital PCR; islets; methylation sensitive restriction enzyme; transplantation; unmethylated insulin DNA; β-cell.

FIGURE 1

Experimental design of the quantification of unmethylated insulin (INS)-DNA using methylation sensitive restriction enzyme (MSRE) digital droplet polymerase chain reaction (ddPCR). (A) DNA is isolated from the samples and subsequently split in two and treated with or without the MSRE. The MSRE cleaves the unmethylated INS-DNA and leaves the methylated INS-DNA intact. (B) The DNA sample is partitioned into thousands of droplets followed by PCR amplification with FAM-labelled hydrolysis probes directed to the INS target DNA and probes directed to a reference gene (HEX-labelled). Droplet reading takes place after amplification. Droplets that are positive or negative for the INS target DNA and/or reference gene are counted to calculate the fraction of unmethylated INS-DNA in the sample. Abbreviations: ddPCR, Digital Droplet polymerase Chain Reaction; INS, Insulin; MSRE, Methylation Sensitive Restriction Enzyme; PCR, polymerase Chain Reaction.

FIGURE 2

Unmethylated INS-DNA fraction in EndoC-βH-1 cells (A–C) and THP-1 cells (D–F) as positive and negative control samples, respectively. The two-dimensional plots from the digital droplet polymerase chain reaction (ddPCR) demonstrate four different clusters each representing different DNA containing droplets: Gray cluster: FAM- HEX-, containing no INS target DNA and no reference copies. Green cluster: FAM- HEX+, containing no INS target DNA but contains reference copies. Blue cluster: FAM+ HEX-, containing INS target DNA but no reference copies. Orange cluster: FAM+ HEX+ containing both INS target DNA and reference copies. The control samples are both split in two and either treated with or without methylation sensitive restriction enzyme (MSRE). The unmethylated INS-DNA fraction is calculated: $1-\frac{\frac{\left[INS\right]}{\begin{array}{c}\left[TTC5\right]\end{array}} with MSRE}{\frac{\begin{array}{c}\left[INS\right]\end{array}}{\left[TTC5\right]} without MSRE }\ast 100\%$ . Abbreviations: ddPCR, Digital Droplet polymerase Chain Reaction; INS, Insulin; MSRE, Methylation Sensitive Restriction Enzyme.

FIGURE 3

A seven point standard curve demonstrates the relation between input percentage of EndoC-βH-1 cells DNA (diluted in a background of THP-1 cells DNA) that could be expected and EndoC-βH-1 cells DNA quantified as unmethylated INS-DNA was measured using digital PCR. (Y = 0.8862 *X + 7.019, r ² = 0.9953, p < 0.0001). Abbreviations: PCR; polymerase Chain Reaction.

FIGURE 4

Unmethylated INS-DNA fraction quantified by digital PCR in different purities of islets, determined via DTZ staining, after isolation from seven donor research pancreases. (r ² = 0.8318, p < 0.0001). Abbreviations: DTZ; Dithizone Staining, PCR; polymerase Chain Reaction.