液滴数字TRAP（ddTRAP）：端粒重复扩增实验方案在液滴数字聚合酶链式反应中的应用

摘要

我们成功将标准的端粒重复扩增方案（TRAP）检测改造为可用于微滴数字聚合酶链式反应。这种新检测方法称为ddTRAP，具有更高的灵敏度和定量能力，可更有效地检测多种人类细胞中的端粒酶活性，并进行更可靠的统计分析。

摘要

端粒重复扩增程序（TRAP）是检测特定样本中端粒酶活性最常用的实验方法。这种基于聚合酶链式反应（PCR）的方法能够对大多数细胞裂解液中的酶活性进行可靠的测定。传统的基于凝胶的TRAP方法使用荧光标记引物，限制了样本通量，且仅能检测酶活性两倍或更大的变化。而液滴数字TRAP（ddTRAP）是一种高灵敏度的方法，由传统TRAP assay改进而来，使用户能够在单次运行中对96个样本进行稳健分析，并实现对每个PCR反应中DNA（即端粒酶延伸产物）输入量的绝对定量。因此，新开发的ddTRAP assay克服了传统凝胶基TRAP assay的局限性，在实验室和临床环境中提供了更高效、更准确和更定量的端粒酶活性检测手段。

引言

端粒是位于线性染色体末端的动态DNA-蛋白质复合物。人类端粒由一系列5'-TTAGGG_n六核苷酸重复序列组成，其长度在出生时约为12–15千碱基（kb）¹。人类端粒酶是一种维持端粒结构的核糖核蛋白酶，最初在HeLa细胞裂解液（癌细胞系）中被发现²。端粒与端粒酶共同在基因组保护、基因调控以及癌细胞永生化等多种生物学过程中发挥重要作用^3,4,5,6。

人类端粒酶主要由两个关键组分构成，即端粒酶逆转录酶和端粒酶RNA（分别为hTERT和hTERC）。其中，蛋白质亚基hTERT是端粒酶中具有催化活性的逆转录酶组分。RNA模板hTERC则为端粒酶提供模板，用于延伸和/或维持端粒。大多数人类体细胞中无法检测到端粒酶活性。由于DNA聚合酶无法延伸DNA滞后链的末端，加之缺乏端粒酶，导致每次细胞分裂后端粒逐渐缩短。这些现象使得大多数体细胞的端粒持续缩短，直至达到临界长度，细胞由此进入复制性衰老状态。细胞可分裂的最大次数由其端粒长度决定，这种对持续分裂的阻断被认为可防止向肿瘤发生发展⁷。癌细胞能够克服端粒诱导的复制性衰老，通过利用端粒酶维持其端粒长度而持续增殖。大约90%的癌症会激活端粒酶，因此端粒酶活性在癌症的检测与治疗中具有至关重要的意义。

20世纪90年代TRAP检测技术的发展对于鉴定端粒酶所必需的组分，以及在多种正常和癌变细胞与组织中测量端粒酶活性起到了关键作用。最初的基于凝胶的PCR检测方法采用放射性标记的DNA底物来检测端粒酶活性。2006年，该检测被改进为使用荧光标记底物的非放射性形式^8,9。通过使用荧光标记的底物，用户可通过将凝胶暴露于适当的激发波长下，观察到端粒酶延伸产物在凝胶上呈现的条带。TRAP检测具有较高的灵敏度，并可在粗制细胞裂解液中检测端粒酶活性，因此成为目前应用最广泛的端粒酶活性检测方法。然而，TRAP检测也存在局限性。该方法依赖凝胶电泳，使得在中高通量研究中难以完成必要的重复实验，因而很少能实现适当的统计学分析。此外，由于无法可靠检测样品间小于两倍的端粒酶活性差异，基于凝胶的检测方法难以进行准确的定量分析。克服上述两项局限性，对于将TRAP等酶活性检测技术应用于临床或工业环境——例如在患者样本分析或药物设计研究中检测端粒酶活性——至关重要。

数字PCR最初于1999年开发，旨在将PCR的指数级模拟特性转化为线性数字化检测方法¹⁰。微滴数字PCR（ddPCR）是原始数字PCR技术的最新发展。随着先进微流控技术和水包油乳液化学的进步，能够稳定生成大小均一的微滴，从而实现了ddPCR。与基于凝胶的方法甚至定量PCR（qPCR）不同，ddPCR可对输入材料进行绝对定量。ddPCR的关键在于通过将样品分割成微滴，生成约20,000个独立的反应。在终点PCR完成后，微滴读取仪以类似流式细胞仪的方式逐个扫描每个微滴，计数、测量大小，并记录每个微滴中荧光的有无（即每个微滴中是否存在PCR扩增子）。随后，利用泊松分布，根据阳性微滴数与总微滴数的比例估算输入分子的数量。该数值代表了每个PCR反应中输入分子数量的估计值。此外，ddPCR在96孔板上进行并完成分析，使用户能够同时运行多个样本，并进行生物学重复和技术重复，以实现适当的统计分析。因此，我们将ddPCR强大的定量能力与中等通量特性相结合，并与TRAP检测技术整合，开发出ddTRAP检测方法¹¹。该检测方法旨在帮助用户研究并稳健地定量来自生物样本的端粒酶绝对活性^11,12。ddTRAP的高灵敏度使其能够从数量有限且珍贵的样本中定量端粒酶活性，包括单细胞水平的测量。此外，用户还可利用该方法以绝对定量方式研究端粒酶调控和/或药物处理的影响，检测小于两倍的变化（约50%的差异）。ddTRAP是TRAP检测技术顺应现代实验室实验和临床环境中数字化与高通量需求的自然演进。

方案

1. Buffer preparation and storage

1. Prepare 50 mL of 1x stock RNase-/DNase-free NP-40 lysis buffer (10 mM Tris-HCl [pH 8.0], 1 mM MgCl₂, 1 mM ethylenediaminetetraacetic acid (EDTA), 1% [vol/vol] NP-40, 10% [vol/vol] glycerol, 150 mM NaCl, 5 mM β-mercaptoethanol, and 0.1 mM 4-benzenesulfonyl fluoride hydrochloride (AEBSF)). This buffer can be aliquoted and stored at -20 °C for future use. Avoid freeze/thaw cycles in order to obtain an optimal lysis of cells.
2. Prepare 50 mL of 10x stock RNase-/DNase-free TRAP extension buffer (200 mM Tris-HCl [pH 8.0], 15 mM MgCl₂, 630 mM KCl, 0.5% [vol/vol] Tween 20, and 10 mM ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA)). This buffer can be aliquoted into 1 mL aliquots and stored at -20 °C for future use.

2. Cell lysis

1. Preparation of the cells
   1. Thaw a frozen aliquot of NP-40 lysis buffer. Once thawed, place the lysis buffer on ice. Add phenylmethylsulfonyl fluoride (PMSF protease inhibitor) to the NP-40 lysis buffer to reach a final concentration of 0.2 mM.
      NOTE: This should be done immediately prior to lysing the cells.
   2. Remove any excess liquid from the collected cell pellets to ensure a dry cell pellet. Note that cell pellets, whether freshly collected or previously frozen (-80 °C or flash frozen in liquid N₂), must not contain any leftover solutions from previous centrifugations as these solutions may interfere with downstream procedures.
   3. Grow, collect, and freeze both telomerase-positive and telomerase-negative cell lines (BJ fibroblasts, IMR-90 or U2OS) in order to use them as positive and negative controls. Use new control lysates every time the assay is performed to ensure assay reproducibility.
      NOTE: It is advised that a large culture of telomerase-negative cells is grown and aliquoted prior to freezing to remove any tissue-culture-related artifacts that may be introduced during the long-term culture of cell lines to help ensure reproducible results of the negative control sample.
   4. Place the cell pellets on ice. If the cells were frozen, allow them to thaw briefly on ice.
      NOTE: Typical cell pellet sizes for the ddTRAP are between 500,000 and 1,000,000 cells. Smaller cell pellets may also be used if necessary, but the cell number must/ideally should be known. The ddTRAP can also be performed based on protein input using a BCA protein assay (input usually is 1 µg).
2. Lysing of the cells
   1. Lyse the cells in the NP-40 lysis buffer. Maintain a cell equivalence of 25,000 cells/µL of buffer. For example, lyse a pellet (containing 1,000,000 cells) in 40 µL of NP-40 lysis buffer. Gently pipet the lysate up and down in order to break open the cells. Try to avoid making bubbles.
   2. Allow the cells to lyse on ice for 30 min. Make sure to gently vortex the lysate to prevent clusters of cell debris from forming. This can be done every 10 min and is meant to keep the lysate a homogenized mixture.
   3. Dilute the cell lysate (25,000 cells/µL) 1:20 in NP-40 lysis buffer, making the new cell equivalence 1,250 cells/µL. For example, dilute 5 µL of cell lysate into 95 µL of lysis buffer.

3. Telomerase extension reaction

1. Prepare a master mix (Table 1) for the telomerase extension reaction (per reaction).
   NOTE: It is best to prepare the extension reaction master mix during the cell lysis and store it on ice.
   1. Pipet 48 µL of extension master mix into each PCR tube.
   2. Add 2 µL of the diluted (1,250 cells/µL) lysate to the extension reaction. The total volume should now be 50 µL with a final cell equivalence of 50 cells/µL.
2. Perform the telomerase extension reaction (Table 2).
3. Store the telomerase extension products at 4 °C for up to 3–5 days; however, it is optimal to use the extension products within the first 24 h.

4. Droplet digital PCR setup

1. Prepare a master mix (Table 3) for the ddTRAP (per reaction).
   NOTE: Once the reagents are at room temperature, do not place them or the master mix on ice. Placing the mix on ice may increase viscosity and lead to poor droplet formation. The DNA polymerase in the ddPCR supermix is a hot start and should be stable at room temperature. The ddPCR supermix may be stored at 4 °C after an initial thaw from -20 °C.
   1. Pipet 19.8 µL of ddPCR master mix into each PCR tube.
   2. Add 2.2 µL of the extension reaction to each tube. Note that the total volume should now be 22 µL.
      NOTE: The total amount of sample needed for a ddTRAP is 20 µL (cell equivalence of 100 cells). The extra volume is a precaution for pipet error or sample loss.
2. Set up the droplet generation cartridge.
   NOTE: The cartridge contains three different columns of wells that are labeled.
   1. Load 20 µL of the reaction prepared according to step 4.1.2 into the sample well (middle well) in the cartridge. Avoid bubbles when pipetting the sample.
      NOTE: If there are bubbles, gently tap the side of the cartridge so that these bubbles come to the top of the solution.
   2. Load 70 µL of droplet generation oil into the oil well (left well).
      NOTE: In order to avoid contamination, strictly use a separate set of pipettes and tips for ddPCR droplet generation steps. The order of loading the cartridge is important. The sample must be loaded prior to loading oil as oil is heavier and will fill the microfluidic chambers and lead to poor droplet formation. The minimum number of samples that can be run is eight. All wells of the cartridge must be loaded with the sample as any empty well will lead to a halt in the droplet generation from the droplet generator. If adequate samples are not available to load all eight wells within a cartridge, 20 µL of ddTRAP master mix (step 4.1) can be loaded into the remaining cartridges.
   3. Secure the gasket in place by tethering it to the ends of the cartridge.
      NOTE: The lack or improper placement of the gasket will prevent the generator from generating droplets.
   4. Place the loaded and assembled cartridge into the droplet generator.
      NOTE: The cartridge is recognized by a magnet in the generator, which will inform the user when the cartridge is placed properly.
3. Remove the cartridge once the droplets are generated and the cycle is complete (~60–90 s).
   1. Gently remove the gasket from the cartridge. Pipet the newly generated droplets (right well), using a multichannel pipette, into a 96-well PCR plate.
      NOTE: The approximate volume for the newly generated droplet emulsion should be 40–43 µL.
   2. Heat seal the plate with aluminum foil PCR plate seals once all the samples are loaded in the 96-well plate in order to prevent evaporation during the PCR steps.
4. Load the 96-well plate into the thermocycler and perform the following PCR reaction (Table 4).
   NOTE: All ramp rates between the temperature steps must be set to 2.5 °C/s in order to properly heat the reactions.

5. Detection of telomerase extension products

1. Load the 96-well plate in the droplet reader.
   NOTE: Make sure to orient the plate properly so that the A1 sample matches with that of the holder.
   1. Open the software associated with the droplet reader. Double-click the first well A1 to open the sample/well editor screen.
   2. Click Experiment and select ABS from the drop-down menu.
      NOTE: Experiment defines the type of assay to be used (i.e., absolute quantification or gene copy number assay). ABS stands for absolute quantification.
   3. Select QX200 ddPCR Evagreen Supermix to ensure that the correct detection method is employed by the reader. Click Apply in the lower right-hand side of the well editor screen to save the user-defined settings to all of the highlighted wells.
      NOTE: SUPERMIX defines the type of PCR mix and detection chemistry that will be read by the reader.
   4. Click on TARGET in the well editor screen of the software in order to define the sample.
   5. Define the TYPE of sample by clicking the TARGET drop-down menu and selecting either unknown, reference, or NTC (no-template control).
      NOTE: Unknown would refer to an experimental sample, reference could be a control sample of known telomerase activity, and an NTC is a critical control for the determination of assay validity in terms of contamination and background signal.
   6. Label all the samples in the Sample Name section and click Apply to ensure the highlighted wells are edited appropriately.
2. Click Run to run/read the plate. Select either Columns or Rows in the RUN OPTIONS screen when prompted to inform the machine about the orientation the plate should be read in.

6. Data analysis

1. Determine the number of accepted droplets for each sample by clicking on individual wells. Double-click the individual wells or column/row headers to view and analyze the sample data.
   NOTE: The most important criteria on whether or not to proceed with the analysis of a particular sample is the number of “accepted droplets”. For the ddTRAP, samples with 10,000 or more accepted droplets are valid for further analysis. Data can be provided to the user in many formats, including a .csv table, .jpg images, histograms, etc. There are many resources available for the in-depth analysis of ddPCR data, including the ddTRAP^11,13. These resources offer a guide to analyzing ddTRAP data, from selecting thresholds^11,13 to choosing samples for further analysis^11,13, identifying false positives and negatives¹³, and general troubleshooting¹³.
2. Highlight the wells representing sample replicates and NTC samples. Analyze the samples in comparison to either NTC or negative control lines, such as BJ cells (as listed above in step 2.1.2).
   1. Manually set the threshold for the samples by clicking on the icon for setting thresholds on the bottom left of the screen. Set thresholds for each individual well or for multiple wells at a time (recommended).
      NOTE: If background is detected in the NTC, it can be subtracted from all of the other samples to ‘normalize’ the signal and ensure that only the true positive signal is analyzed. NTC values are typically in the 0.2–1 molecule/µL range for the NP-40 lysis buffer system. Other buffer systems and components would need to be tested (for example, CHAPS buffers).

结果

利用ddTRAP，在包含以下细胞系的细胞组中检测了端粒酶活性（图1）：非小细胞肺癌（H2882、H1299、Calu6、H920、A549 和 H2887）、小细胞肺癌（H82 和 SHP77）以及端粒酶阴性的成纤维细胞（BJ）。每组取一百万个细胞沉淀，用NP-40裂解缓冲液裂解，并进行生物学重复三次的端粒酶延伸反应。一个常用且高度推荐的阴性对照是“NTC”（无模板对照）。该样本通过向端粒酶延伸反应体系中加入NP-40裂解缓冲液（2 µL）制备，并与其他含实际细胞裂解液的样本以完全相同的方式处理延伸产物。该样本可帮助使用者扣除可能存在的背景信号，从而更准确地定量端粒酶活性。尽管本图中未展示，另一种阴性对照方法是在端粒酶延伸反应前将裂解液在95°C下加热失活5分钟。若细胞/样本量充足，建议优先采用此阴性对照方法。

通过测量液滴乳液中每个液滴的荧光强度，液滴读取仪可利用泊松分布估算输入分子的浓度（...

讨论

端粒酶活性的测定对于众多研究领域至关重要，包括但不限于癌症、端粒生物学、衰老、再生医学以及基于结构的药物设计。即使在癌细胞中，端粒酶核糖核蛋白复合物（telomerase RNPs）的丰度也很低，这使得该酶的检测与研究具有挑战性。本文中，我们详细描述了新开发的数字液滴式TRAP（ddTRAP）检测技术的逐步操作流程，以可靠地定量细胞中的端粒酶活性。通过将传统的端粒酶延伸反应与数字液滴PCR（ddPCR）相结合，我们能够在肺癌细胞中定量检测端粒酶活性（即端粒酶延伸产物）。

ddTRAP 检测基于与 TRAP 检测相同的原理。通过在非离子型去污剂（NP-40）裂解缓冲液中裂解细胞获得细胞裂解物，以保持端粒酶活性，随后用于对“TS”底物/引物进行端粒酶延伸反应。ddTRAP 的创新之处在于 PCR 前形成微滴。将样本分割成微滴可实现对每个细胞中端粒酶活性的绝对定量。

使用数字液滴式TRAP（ddTRAP）检测了肺癌细胞系中的端粒酶活性。基于凝胶的TRAP检测方法的一个主要局限性是单次可处理的样本数量。大多数凝胶梳子最多只能容纳20个...

材料

本文使用的材料清单

姓名	公司	目录编号	评论
1 M Tris-HCl pH 8.0	Ambion	AM9855G	无RNA酶/DNA酶
1 M MgCl2	Ambion	AM9530G	无RNA酶/DNA酶
0.5 M EDTA pH 8.0	Ambion	AM9261	无RNA酶/DNA酶
Surfact- Amps NP-40	Thermo Scientific	28324
100% 超纯甘油	Invitrogen	15514011	无RNA酶/DNA酶
苯甲基磺酰氟	Thermo Scientific	36978	粉末
2-巯基乙醇	SIGMA-ALDRICH	516732
无核酸酶水（Nuclease Free H20）	Ambion	AM9932	无RNA酶/DNA酶
2.5 mM dNTP 混合液	Thermo Scientific	R72501	每种dATP、dCTP、dGTP和dTTP各2.5 mM
2 M KCl	Ambion	AM9640G	无RNA酶/DNA酶
100% Tween-20	Fisher	9005-64-5
0.5 M EGTA pH 8.0	Fisher	50-255-956	无RNA酶/DNA酶
端粒酶底物（TS）引物	Integrated DNA Technology (IDT)	定制引物（HPLC纯化）	5'- AATCCGTCGAGCAGAGTT-3'
ACX（反向）引物	Integrated DNA Technology (IDT)	定制引物（HPLC纯化）	5'- GCGCGGCTTACCCTTACCCTTACCCTAACC -3'
薄壁（250 µl）PCR级管	USA Scientific	1402-2900	条板、板、管等
QX200 ddPCR EvaGreen Supermix	Bio Rad	1864034
Twin-Tec 96孔板	Fisher	Eppendorf 951020362
可穿刺铝箔热封膜	Bio Rad	1814040
液滴生成器芯片（DG8）	Bio Rad	1863008
液滴生成油	Bio Rad	1863005
液滴生成器垫圈	Bio Rad	1863009
96孔热循环仪 T100	Bio Rad	1861096
PX1 PCR板封膜机	Bio Rad	1814000
QX200 液滴读取仪及Quantasoft软件	Bio Rad	1864001 和 1864003
ddPCR液滴读取仪用油	Bio Rad	1863004
无核酸酶滤芯吸头	Thermo Scientific		10 µl、20 µl、200 µl 和 1000 µl