差异甲基化的测量<em> INS</em> DNA种人血清中为胰岛生物标志物β细胞死亡
Summary
Islet β cell death precedes development of type 1 diabetes, and detecting this process may allow for early therapeutic intervention. Here, we provide a detailed description of how to measure differentially methylated INS DNA species in human serum as a biomarker of β cell death.
Abstract
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.
Introduction
1型糖尿病（T1D）是一种自身免疫性疾病是受自身反应性T细胞1特征在于胰岛素产生胰岛β细胞的破坏。 T1D的诊断通常是在贫，年轻个体，谁可能酮症酸中毒表现为胰岛素缺乏的证据后高血糖症（血糖> 200mg / dl的）测量的。在T1D的诊断时，存在用于β细胞功能和质量的显着损失的证据（50 – 90％）2。在临床研究中，在诊断时被提起的几个免疫调节药物导致β细胞功能（大概质量），但没有的稳定已导致疾病的临床缓解，已提出的呼叫为发展的裁断生物标志物为早期检测疾病的和组合的有效性的纵向跟踪治法3,4。由国际财团的努力，这样的S中的人胰岛研究网络协会在希思5全国学院，都强调了需要开发侧重于T1Dβ细胞应激和死亡的生物标志物。
在这些努力线，我们组和其他人最近开发了测量循环，从垂死的β细胞主要6出现表观遗传学修饰的DNA片段的生物标志物检测– 9。在所有的公布分析迄今，重点是对人类基因编码前胰岛素原（INS），这表明更高的程度相比其他细胞类型的非甲基化CpG部位中的编码和启动子区域的定量。未甲基化的INS DNA片段的解放被假设为主要从死亡线上（坏死，凋亡）β细胞发生。我们最近的研究表明，青少年，在这两个非甲基化和甲基化DNA INS海拔在位置-69基点（相到t他转录起始位点），新发1型糖尿病进行观察，并一起担任特异性标志物为这个人口6。这些生物标志物测定涉及使用商业自旋试剂盒的血清或血浆的无细胞的DNA的分离，接着是分离的DNA的亚硫酸氢盐转化率（以非甲基化胞嘧啶转换为尿嘧啶，留下甲基化的胞嘧啶完整）。
在这份报告中，我们描述了血清样品的采集，从血清中游离DNA，亚硫酸氢盐转化和滴数字PCR（下文，数字PCR）的性能隔离的差异甲基化DNA INS技术方面。
Protocol
Representative Results
Discussion
通过DNA甲基化的胞嘧啶甲基化使得转录在许多基因的表观遗传调控。在人体中的INS基因在胰岛β细胞几乎完全表达，似乎有在INS基因胞嘧啶甲基化的频率之间的相关性，以它的转录11的沉默。因此，大多数细胞类型显示在较β细胞11多种胞嘧啶的INS基因的甲基化相当高的频率– 13。已经提出了β细胞死亡的发生率可通过无细胞未甲基化的INS DN…
Disclosures
The authors have nothing to disclose.
Acknowledgements
This work was supported by National Institutes of Health grant UC4 DK104166 (to RGM). We wish to acknowledge the assistance of the Indiana Diabetes Research Center Translation Core supported by National Institutes of Health grant P30 DK097512.
Materials
| Red Top Vacutainer | Beckon Dickinson | 366441 | no additive, uncoated interior, 10 ml |
| Cryovial Tube | Simport | T310-3A | polypropylene, screw cap tube, any size |
| QIAamp DNA Blood Mini Kit | Qiagen | 51106 | |
| Poly(A) | Sigma | P9403 | disloved in TE buffer (10 mM Tris-Cl pH 8.0 + 1 mM EDTA) to 5 µg/µl |
| Absolute Ethanol (200 Proof) | Fisher Scientific | BP2818-500 | |
| DPBS (with CaCl and MgCl) | Sigma | D8662 | |
| 0.2 mL PCR 8-strip Tubes | MidSci | AVST | |
| 8-strip Caps, Dome | MidSci | AVSTC-N | |
| EZ DNA Methylation-Lightning Kit | Zymo | D5031 | |
| ddPCR Supermix for Probes (No dUTP) | Biorad | 1863024 | |
| Buffer Control for Probes | Biorad | 1863052 | |
| Human Unmethylated/Methylated Primer/Probe mix | Life Technologies | AH21BH1 | |
| EcoR1 | New England Biolabs | R0101L | |
| twin.tec PCR Plate 96, semi-skirted | Eppendorf | 951020346 | |
| Pierceable Foil Heat Seal | Biorad | 1814040 | |
| PX1 PCR Plate Sealer | Biorad | 1814000 | |
| QX200 AutoDG Droplet Digital PCR System | Biorad | 1864101 | |
| Automated Droplet Generation Oil for Probes | Biorad | 186-4110 | |
| DG32 Cartridge for Automated Droplet Generator | Biorad | 186-4108 | |
| Pipet Tips for Automated Droplet Generator | Biorad | 186-4120 | |
| Pipet Tip Bins for Automated Droplet Generator | Biorad | 186-4125 | |
| C1000 Touch Thermal Cycler | Biorad | 1851197 | |
| QX200 Droplet Reader | Biorad | 186-4003 | |
| ddPCR Droplet Reader Oil | Biorad | 186-3004 |
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