Tersey Sarah A, Nelson Jennifer B, Fisher Marisa M, Mirmira Raghavendra G
Department of Pediatrics, IU Center for Diabetes and Metabolic Disease, Indiana University School of Medicine.
Department of Pediartics, Omaha Children's Hospital and Medical Center, University of Nebraska Medical Center.
J Vis Exp. 2016 Dec 21(118):54838. doi: 10.3791/54838.
The 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.
胰岛β细胞的死亡被认为是几乎所有形式糖尿病发病机制的基础，且早于明显高血糖的出现，尤其是在1型糖尿病中。开发敏感且可靠的β细胞死亡生物标志物可能有助于早期进行治疗干预，以预防或延缓糖尿病的发展。最近，包括我们自己的几个研究小组报告称，循环中编码胰岛素原（INS）的无细胞、差异甲基化DNA与1型糖尿病前期和新发1型糖尿病中的β细胞死亡相关。在这里，我们展示了一种使用数字PCR测量无细胞INS DNA的分步方案，该DNA在相对于转录起始位点的-69 bp处的胞嘧啶存在差异甲基化。我们证明该检测方法能够区分-69 bp处甲基化和未甲基化的胞嘧啶，在几个数量级上呈线性关系，可提供DNA拷贝数的绝对定量，并且可应用于新发1型糖尿病患者和无病对照的人血清样本。本文所述方案可适用于任何需要检测差异甲基化胞嘧啶的DNA种类，无论是来自循环系统还是分离的细胞和组织，并且能够提供DNA片段的绝对定量。
