差异甲基化的测量<em&gt; INS</em&gt; DNA种人血清中为胰岛生物标志物β细胞死亡

摘要

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.

摘要

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.

引言

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。

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研究方案

Ethics Statement: Protocols were approved by the Indiana University Institutional Review Board. Parents of subjects provided written informed consent, and children older than 7 years provided assent for their participation.

1. Serum Processing

NOTE: The assay as described has been rigorously tested using human serum isolated as follows.

1. Collect blood in one red top (no-additive; uncoated) blood collection tube.
2. Let sit at room temperature for 30 min to allow the clot to form.
3. Centrifuge tube at 2,000 x g for 10 min at room temperature. Transfer the supernatant (serum) to a new tube designated for serum storage (polypropylene screw top tubes, size dependent on samples).
4. Store samples at -80 ºC until ready for DNA isolation.

2. Serum DNA Extraction

NOTE: DNA is extracted with a DNA extraction kit using 50 µl of serum (recommended), following manufacturer's protocol with some modifications.

1. Prepare 200 µl lysis buffer + 1 µl poly(A) (5 µg) per sample in an microcentrifuge tube. Vortex for 15 sec. Set aside until Step 2.3.
2. Take serum samples from -80 ºC freezer and thaw at room temperature. Bring serum sample volume up to 200 µl total using phosphate-buffered saline (PBS, with calcium chloride and magnesium chloride).
3. Lyse the sample by adding 20 µl protease (1.4 Anson units/ml) to sample, followed by 200 µl lysis buffer/polyA mix (from Step 2.2) and vortex for 8 sec. Incubate sample at 56 °C for 10 min, then briefly centrifuge for 7 sec at top speed in a microcentrifuge (>10,000 x g).
4. Precipitate DNA by adding 230 µl 100% ethanol to sample and vortex for 8 sec. Briefly centrifuge for 7 sec at top speed (>10,000 x g).
5. Apply the DNA to the spin column by adding 600 µl sample mixture to mini spin column. Centrifuge at 6,000 x g for 1 min. Discard the flow-through and place column in a clean tube.
6. Wash the DNA by adding 500 µl wash buffer 1 to the column. Centrifuge at 6,000 x g for 1 min. Discard the flow-through and place column in a clean tube. Add 500 µl wash buffer 2 to column and centrifuge at top speed (>10,000 x g) for 3 min. Place column in a new 1.5 ml microcentrifuge tube and centrifuge at top speed again for 1 min.
7. Elute the DNA by placing column in a new 1.5 ml microcentrifuge tube and adding 60 µl elution buffer directly on the filter. It is important to switch tips between samples to prevent cross contamination. Incubate at room temperature for 5 min, then centrifuge at 6,000 x g for 1 min. Add another 60 μl of elution buffer and centrifuge at 6,000 x g for 1 min. 
8. Store DNA at -20 ºC for later use, or proceed immediately to bisulfite conversion.

3. Bisulfite Conversion

NOTE: Bisulfite conversion is performed using a bisulfite conversion kit, following the manufacturer's protocol with some modifications.

1. To convert unmethylated cytosines to uracils, add 130 µl of bisulfite conversion reagent to 20 µl of DNA from step 2.7 in a PCR tube (single 0.2 ml PCR tube or 8-strip PCR tubes). Save the remaining 40 µl of DNA for future use, or use in replicate reactions. Mix 20 times by pipetting up and down and centrifuge briefly to make sure no drops are on sides or lid. Incubate in a water bath or thermal cycler as follows: 98 °C for 8 min, 54 °C for 60 min, 4 °C for 5 min.
   1. Proceed to step 3.2 or store at 4 °C for up to 20 hr.
2. Add 600 µl of binding buffer to spin column and place the column in the provided collection tube. Apply the bisulfited DNA to column and mix by pipetting up and down 10 times. Centrifuge at top speed (>10,000 x g) for 30 sec. Discard flow-through.
3. Wash DNA by adding 100 µl of wash buffer to column and centrifuge at top speed (>10,000 x g) for 30 sec.
4. Perform desulphonation to remove sulphonate group to finalize the conversion of unmethylated cytosines to uracil by adding 200 µl of desulphonation buffer to the column. Let stand at room temperature for 20 min, then centrifuge at top speed (>10,000 x g) for 30 sec.
5. Wash DNA by adding 200 µl of wash buffer to the column and centrifuge at full speed for 30 sec. Discard the flow-through and repeat this wash step.
6. Place the column into a 1.5 ml microcentrifuge tube. Elute DNA by adding 10 µl elution buffer directly to column filter. Incubate for 1 min. Centrifuge for 30 s at full speed to elute DNA. Quantify recovered DNA by spectrophotometry at A260.
   NOTE: The DNA detected by spectrophotometry represents primarily the carrier polyA DNA that was added at step 2.3 above. Typically, recovery of polyA is ≥85% of the input (>4.25 µg).
7. Store DNA at -20 °C until ready to proceed with digital PCR.

4. Multiplex Digital PCR

1. Make a master mix with enough solution for each sample and control. Include at least one sample containing water (negative control), one sample of a plasmid containing unmethylated INS (1 pg, positive control), one sample of plasmid containing methylated INS (1 pg, positive control), and one sample containing a 1:1 mixture of plasmids.
   1. To prepare the PCR master mix, add 12.5 µl per reaction of PCR buffer (e.g., 125 µl per 10 samples), 1.25 µl per reaction primer/probe mix (e.g., 12.5 µl per 10 samples), 8.25 µl per reaction water (e.g., 82.5 µl per 10 samples), 0.5 µl per reaction EcoR1 enzyme (e.g., 5 µl per 10 samples).
      NOTE: Primers and probes used here are as follows: Forward Primer: 5'-GGAAATTGTAGTTTTAGTTTTTAGTTATTTGT-3'; Reverse Primer: 5'-AAAACCCATCTCCCCTACCTATCA-3'; FAM probe: FAM-5'-ACCCCTACCACCTAAC-3'-MGB; VIC probe: VIC-5'-ACCCCTACCGCCTAAC-3'-MGB.
2. Set up multiplex PCR reaction in a 96-well plate. Add 19.5 µl of master mix to each well. Add 2.5 µl of bisulfite converted DNA sample into each appropriate well (save the remaining 7.5 µl of bisulfite converted DNA or use in replicate reactions). Mix by pipetting up and down several times.
   NOTE: All 8 wells in a row must contain sample or buffer control.
3. Seal with a foil seal using a plate sealer and centrifuge in a plate spinner until there is no liquid on the sides of the wells.
4. Set up the automated droplet generator on the touchscreen.
   NOTE: All 8 wells in the cartridge must contain sample or buffer control.
   1. Set the number of rows being used by touching the row(s) in which samples are loaded into on the 96-well plate from step 4.2 and highlight the row in blue to indicate an active row.
   2. Load consumables from back to front to avoid contamination. To start, add cartridges along the back row of the instrument. Load tips into center row of the instrument.
      NOTE: The cartridges can only fit into the holders in the correct orientation.
   3. Place 96-well plate into the instrument. Remove a cold block from -20 °C freezer and place a new skirted 96-well plate into it. Place it inside the instrument next to the sample loaded 96-well plate.
   4. Add oil to dispenser in the front of the instrument. Select type of oil on touchscreen.
   5. Touch the blue start button to start the run. Confirm plate setup and touch start run button to begin.
5. Once finished, remove the 96-well plate that contains the newly formed droplets and seal with a foil seal using a plate sealer.
6. Perform PCR₁₀ on a thermal cycler using the following program: 95 °C for 10:00 min; 40 cycles at: 94 °C for 00:30 min, 57.5 °C for 01:00 min; 98 °C for 10:00 min; 12 °C for 10 min or up to 24 hr.
7. Place the plate on the droplet reader and set up the reader.
   1. Click on the setup tab. Open a new template by clicking template > new. Enter a file name.
   2. Use the well editor to set the parameters for each sample. Give each sample a unique name, set experiment to Rare Event Detection (R-RED), and set master mix to the mix used in step 4.1.
   3. Give Target 1 a name (i.e., unmethylated) and set Target 1 as unknown (U). Give Target 2 a name (i.e., methylated) and set Target 2 as reference (R).
   4. Click Run tab to start the run. In the run options window, select the detection chemistry (FAM/VIC).

5. Data Analysis

1. Open results in analyze tab and analyze using 2-D plot based on positive controls (see Figure 1).
2. Export CSV file generated by droplet reader to a spreadsheet program for data analysis.
3. To generate "copies per µl" of serum, use the formula: (concentration*250)/(volume of serum used for DNA isolation in µl).⁶
   NOTE: The data is typically converted to log₁₀ when acquired from human serum to ensure normal distribution. Alternatively, data can be plotted on a log₁₀ scale and analyzed by non-parametric statistics.

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结果

为了正确地解释数据，我们使用质粒控制在每个数字PCR同时运行非甲基化和甲基化的目标DNA INS。这些控件确保对应于甲基化和未甲基化的DNA的信号是清晰可辨。 图1示出对应于液滴对含亚硫酸氢盐转化的未甲基化的INS的DNA（ 图1A）质粒对照，甲基化的INS的DNA（ 图1B）的2-D散点图，并以1:两种质粒（ 图1C）

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讨论

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...

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材料

Name	Company	Catalog Number	Comments
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 CaCl2 and MgCl2)	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