我们要感谢Prachi Shah女士最初帮助我们优化协议。我们要感谢Manoj Garg博士提供A2780卵巢癌细胞系。EK得到了生物技术系（编号BT / RLF / Re-entry/06 / 2015），科学和技术部（ECR / 2018 / 002117）和NMIMS种子基金（IO 401405）的研究资助。

注意：有关以下方案中使用的所有试剂的详细信息，请参阅 材料表 。表 1 列出了实验室制造的试剂以及优化的体积， 表2 显示了市售试剂的工作浓度。
1. 细胞培养
<ol><li>在 Dulbecco 的改良鹰培养基 （DMEM） 完全培养基中维持要测量端粒长度的细胞（此处使用的是 A2780 细胞，这是一种卵巢腺癌细胞系），该培养基补充有 10% 胎牛血清 （FBS）、链霉素、青霉素和两性霉素 B 在 6 cm 培养皿中。在含有5%二氧化碳的加湿和受控环境中于37°C孵育，直到细胞80%-100%汇合。</li>
	<li>取出培养基并用 5 mL 的 1x 磷酸盐缓冲盐水 （PBS） 洗涤。</li>
	<li>用1mL胰蛋白酶 - 乙二胺四乙酸（EDTA）处理细胞，通过在37°C孵育3-5分钟来分离细胞。</li>
	<li>加入 2 mL DMEM 完全培养基以灭活胰蛋白酶并将细胞收集在离心管中。</li>
	<li>以2，348× g 沉淀细胞5分钟。</li>
	<li>用1x PBS洗涤沉淀，并以2，348× g 离心5分钟。</li>
	<li>将颗粒储存在-80°C直至进一步使用。 注意：细胞计数可能因细胞系而异。在A2780细胞系的情况下，我们获得了大约2.5×106 个细胞，用于进一步的步骤。</li>
</ol>2. 基因组DNA分离
<ol><li>向细胞沉淀中加入 500 μL 裂解缓冲液（10 mM tris-Cl，pH 8.0;25 mM EDTA，pH 8.0;100 mM NaCl;0.5% w/v 十二烷基硫酸钠），并使用切割尖端（开口直径至少为 2 mm）轻轻混合。加入 20 μg/mL 新鲜制备的 RNase A，并通过倒置轻轻混合。</li>
	<li>在37°C孵育30分钟，并在孵育过程中偶尔倒置管。</li>
	<li>加入蛋白酶K至终浓度为100μg/ mL，并通过倒置轻轻混合10次。在55°C孵育2小时。在孵育期间定期（每10分钟）倒置管。</li>
	<li>加入 500 μL 苯酚：氯仿：异戊醇试剂 （25：24：1），倒置轻轻混合 20 次。在室温（约25°C）下以9，391 ×g 离心15分钟。 注意： 图1A 显示了离心后获得的三层。</li>
	<li>从可见的三层中取出粘稠的上层水层，使用切口放入新管中，并加入等量的氯仿。轻轻倒置混合20次。</li>
	<li>在室温下以9，391 ×g 离心管15分钟。</li>
	<li>收集水层并加入适量的5M NaCl，使NaCl的最终浓度为0.2 M。</li>
	<li>加入两体积的100%乙醇。轻轻倒置混合20-25次。在室温下以15，871× g 离心5分钟。</li>
	<li>除去上清液并加入 500 μL 70% 乙醇以洗涤沉淀。在室温下以15，871× g 离心5分钟。</li>
	<li>除去上清液，风干沉淀几分钟，然后加入 50 μL 无菌无核酸酶水。让DNA在室温下再水化1-2天。使用切头移液混合。</li>
	<li>使用紫外（UV）分光光度法测量DNA的浓度。如有必要，使用无菌无核酸酶水重新稀释样品，以获得最低浓度为 300-500 ng/μL。</li>
	<li>通过在1%琼脂糖凝胶上运行来检查DNA的完整性（图1B）。</li>
	<li>将稀释的DNA储存在-20°C直至进一步使用。</li>
</ol>3. 基因组DNA的消化
<ol><li>制备Rsa1（20U）和Hinf1（20U）的酶混合物，每次反应加入2μL限制性酶切缓冲液。</li>
	<li>取适当体积的基因组DNA，使DNA总量为1.5μg。用无菌无核酸酶的水补足体积，使酶加入后的总体积为20μL。</li>
	<li>通过敲击混合均匀，然后脉冲旋转。</li>
	<li>将混合物在37°C孵育2小时。</li>
</ol>4. 琼脂糖凝胶电泳
<ol><li>使用高级琼脂糖在 1x 三乙酸酯 EDTA （TAE） 缓冲液中制备 10 cm × 15 cm 0.8% 琼脂糖凝胶。</li>
	<li>向每个消化的基因组 DNA 样品中加入 5 μL 上样染料，从而使最终体积为 25 μL，并将样品上样到凝胶上。</li>
	<li>使用 1 μL 分子标记物（分子量标准）、3 μL 无菌无核酸酶水和 1 μL 5x 上样染料制备分子标记混合物。</li>
	<li>如果样品数量较多（~10），则在基因组DNA消化样品的两侧加载5 μL分子标记混合物，如果样品少于或等于5个，则仅在一侧加载。</li>
	<li>以5 V / cm运行凝胶6小时。电泳完成后，在凝胶的一角做一个凹口以标记上样顺序。</li>
	<li>将凝胶浸入0.25M HCl中，在室温下轻轻搅拌10分钟。</li>
	<li>用蒸馏水冲洗凝胶两次。</li>
	<li>通过将凝胶浸入0.5M NaOH和1.5M NaCl的溶液中两次，每次15分钟，在室温下轻轻搅拌，使DNA变性。</li>
	<li>用蒸馏水冲洗凝胶两次。</li>
	<li>通过将凝胶浸入 0.5 M tris-HCl 和 3 M NaCl （pH 7.5） 的溶液中两次，在室温下轻轻搅拌 15 分钟来中和 DNA。</li>
</ol>5. 南方印迹
<ol><li>切下10厘米×12厘米大小的尼龙膜，并在与凝胶相同的位置做一个缺口。</li>
	<li>通过浸入蒸馏水，然后浸入20x盐水柠檬酸钠（SSC）缓冲液来激活膜（见 表1）。</li>
	<li>设置转移。<ol><li>拿一个干净的玻璃托盘，将另一个托盘倒置放入其中。使用普通滤纸制作灯芯，使两端接触外托盘的底部。</li>
		<li>将凝胶放在滤纸上。轻轻地将活化的尼龙膜放在凝胶上，凹口重叠，并通过滚动玻璃棒去除任何气泡。</li>
		<li>将 2 厘米的 9.5 厘米堆× 11.5 厘米纤维素滤纸，然后放入一堆 6 厘米相同尺寸的普通滤纸。在此设置顶部放置一个砝码，以便在下方具有相等的权重分布。用20x SSC缓冲液填充外托盘（见 图2）。</li>
		<li>将设置留给隔夜转移。</li>
	</ol></li>
	<li>南方转移后，通过在紫外透射仪或紫外交联器上将转移的DNA固定在膜上。使用 302 nm 8 W 灯 5 分钟，或 254 nm 8 W 灯 2 分钟，相当于大约 120 mJ。确保转移DNA的膜侧面向灯。</li>
	<li>用 25 mL 的 2x SSC 缓冲液洗涤膜两次。</li>
	<li>如果需要，通过完全干燥印迹并将其储存在2-8°C后将其松散地包裹在箔纸中来暂停方案，直到进一步处理。</li>
</ol>6. 杂交和化学发光检测
<ol><li>将预杂交缓冲液预热至42°C。 注意：以下步骤包括每100 cm2 印迹膜要使用的溶液/缓冲液体积。</li>
	<li>将膜在10mL预杂交缓冲液中在42°C下孵育1小时，轻轻搅拌以进行预杂交。</li>
	<li>每 5 mL 预热预杂交缓冲液加入 0.5 μL 端粒探针，制成杂交溶液。</li>
	<li>将印迹在42°C的10mL杂交溶液中孵育3小时，轻轻搅拌。</li>
	<li>在室温下用严格的缓冲液1（表1）洗涤印迹两次，每次10分钟（每次25mL）。</li>
	<li>在50°C下预热严格的缓冲液2（表1）30分钟。</li>
	<li>用严格的缓冲液2洗涤印迹两次，在50°C下轻轻搅拌15分钟（每次25mL）。</li>
	<li>用 15 mL 洗涤缓冲液冲洗 5 分钟，在室温下轻轻搅拌。</li>
	<li>将膜在室温下在10mL新鲜制备的1x封闭溶液中孵育30分钟，轻轻搅拌。</li>
	<li>将膜在10mL与碱性磷酸酶工作溶液（见 表2）偶联的抗地灰辛中孵育30分钟，并在室温下轻轻搅拌。</li>
	<li>用洗涤缓冲液在室温下轻轻搅拌两次印迹15分钟。</li>
	<li>在室温下在 10 mL 检测缓冲液中孵育 5 分钟，轻轻搅拌。</li>
	<li>去除多余的检测缓冲液，并在杂交袋或乙酸盐片上保持DNA朝上的膜。将~1-1.5mL底物溶液滴加到膜上，立即将另一张纸放在其上，并在室温下孵育5分钟。</li>
	<li>挤出多余的底物溶液进行成像。</li>
	<li>在凝胶成像系统中对印迹膜成像约20分钟，在不同时间点收集多个图像。选择不饱和的图像进行进一步分析。 注意：如果信号较弱，可以进行更长的成像时间。</li>
</ol>7. 分析
<ol><li>获取图像文件后，以尽可能高的分辨率（在本例中为 600 dpi）以.tif格式导出它们以供发布。</li>
	<li>安装 端工具 软件。这需要运行特定版本的 MATLAB（免费提供）（R2012a）。</li>
	<li>打开软件，然后单击右上角的 “加载图像文件” 按钮。</li>
	<li>加载图像后，单击反转图像，使 图像背景为黑色，拖尾/条带为白色。</li>
	<li>从孔开始裁剪顶角的图像。选择裁剪后，右键单击其中并选择 裁剪图像。</li>
	<li>裁剪图像后，单击计算车道并允许自动进行 车道 检测。</li>
	<li>如果未正确检测到车道，例如，根本没有检测到某些车道，或者检测到额外或部分车道，请按如下所述执行车道调整。<ol><li>单击调整车道，然后在打开的弹出窗口中根据需要添加和/或调整 车道 ，然后单击 应用并关闭。</li>
	</ol></li>
	<li>调整车道后，确保在每个车道上看到一个红点，并使用梯子 拟合 按钮调整梯子，确保两个梯形车道中的峰数位于同一位置。使用 “删除极值 ”按钮删除任何无关的峰值。</li>
	<li>调整梯形图后，选择“ 车道剖面图”，单击 “梯形拟合”，然后选择“多项 式拟合”。</li>
	<li>单击软件建议的趋势 线，然后在下一个选项中选择 更正 。</li>
	<li>单击 “获取 结果”以表格形式显示结果，其中 平均 TRF 行是相应泳道样本的端粒长度测量值。</li>
	<li>单击 全部保存 以电子表格的形式存储结果。</li>
</ol>

使用非放射性化学发光检测定量端粒长度

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作者没有利益冲突。

我们描述了一种使用南方印迹法测量端粒长度的非放射性、基于化学发光的方法的详细程序。该方案已经过测试，允许明智地使用多种试剂，而不会影响结果质量。预杂交和杂交缓冲液最多可重复使用五次。酶浓度可以在每1.5-2μg基因组DNA的10-20U之间变化，而不会影响结果。其他几种试剂盒组分，如DIG标记的分子量标记物和杂交探针，可以在低于推荐浓度的浓度下使用。优化的卷如 表 2</stro...

端粒是存在于染色体末端的重复DNA序列。它们具有TTAGGG的串联重复，并通过保护染色体免受磨损和末端复制问题来维持基因组完整性，这意味着3'突出的部分无法被DNA聚合酶1，2复制。短端粒导致细胞染色体异常，因此细胞在称为复制衰老3的阶段永久停滞。短端粒还会导致许多其他问题，例如线粒体功能障碍4，5 和细胞功能障碍。
当细胞分裂时，DNA端粒重复序列丢失，平均每年损失25至200 bp6，导致细胞在一定次数的分裂后衰老6。衰老与合并症的频率较高有关，其特征是端粒长度缩短7。正如Mender所描述的端粒限制性片段（TRF）分析是一种非常昂贵的方法8。因此，在大多数研究中，在量化端粒长度时没有实施。
目前，大多数流行病学研究采用基于定量聚合酶链反应（qPCR）的端粒长度测量。然而，基于qPCR的方法是一种相对测量方法，因为它测量端粒和单拷贝基因扩增产物之间的比率，而不是绝对端粒长度。使用 TRF 协议测量端粒长度是金标准方法，因为它可以测量样品中的端粒长度分布，并且测量值可以用以千碱基 （kb） 为单位的绝对值表示。然而，它的使用是有限的，因为它繁琐、劳动密集型且成本高昂。在这里，我们提出了一种使用基于化学发光的TRF测量端粒长度的优化协议。
TRF 分析包括七个主要步骤：1） 培养用于基因组 DNA 提取的细胞，2） 使用苯酚：氯仿：异戊醇 （P：C：I） 方法进行基因组 DNA 提取，3） 基因组 DNA 的限制性酶切，4） 琼脂糖凝胶电泳，5） 限制性酶切 DNA 片段的南部印迹，6） 杂 交和检测化学发光 - 固定化端粒探针通过碱性磷酸酶的高灵敏度化学发光底物可视化，2-氯-5-（4-甲氧基螺[1，2-二氧杂环丁烷-3，2′-（5-氯三环[3.3.1.13.7]癸烷]）-4-基]-1-苯基磷酸酯（CDP-Star）-和7）分析，以从这些端粒涂片中获得平均端粒长度和范围信息。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Cell Line</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>A2780 (Ovarian adenocarcinoma cell line)</td>
			<td>Received as a gift</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ChemiDoc XRS+ (for imaging and UV cross linking)</td>
			<td>Biorad</td>
			<td>Universal hood II (721BR14277)</td>
			<td></td>
		</tr>
		<tr>
			<td>Nanodrop (Epoch 2)</td>
			<td>Biotek</td>
			<td>EPOCH2</td>
			<td></td>
		</tr>
		<tr>
			<td>Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>TeloTool</td>
			<td>Version 1.3</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Acetic Acid</td>
			<td>Molychem</td>
			<td>64-19-7</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>MP</td>
			<td>180720</td>
			<td></td>
		</tr>
		<tr>
			<td>Amphotericin B</td>
			<td>Gibco, ThermoFisher Scientific, USA</td>
			<td>15240062</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM&#160;</td>
			<td>HyClone, Cytiva, USA</td>
			<td>SH30243.01</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylenediamine tetraacetic acid&#160;</td>
			<td>Molychem</td>
			<td>6381-92-6</td>
			<td></td>
		</tr>
		<tr>
			<td>HI FBS</td>
			<td>Gibco, ThermoFisher Scientific, USA</td>
			<td>10270106</td>
			<td></td>
		</tr>
		<tr>
			<td>HCl</td>
			<td>Molychem</td>
			<td>76-47-01-0</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Molychem</td>
			<td>7647-14-5</td>
			<td></td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>Molychem</td>
			<td>1310-73-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Nylon membrane</td>
			<td>Sigma</td>
			<td>11209299001</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin</td>
			<td>Gibco, ThermoFisher Scientific, USA</td>
			<td>15240062</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium dodecyl sulfate</td>
			<td>Affymetrix</td>
			<td>151-21-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptomycin</td>
			<td>Gibco, ThermoFisher Scientific, USA</td>
			<td>15240062</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris</td>
			<td>BIORAD</td>
			<td>77-86-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris HCl</td>
			<td>Sigma Aldrich</td>
			<td>1185-53-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Whatman paper</td>
			<td>GE healthcare lifesciences</td>
			<td>1001-917</td>
			<td></td>
		</tr>
		<tr>
			<td>Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1 kb ladder</td>
			<td>NEB</td>
			<td>N3232S</td>
			<td></td>
		</tr>
		<tr>
			<td>20x SSC</td>
			<td>Invitrogen</td>
			<td>15557-036</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti DIG AP</td>
			<td>Telo TAGGG Telomere Length Assay kit</td>
			<td>12209136001</td>
			<td></td>
		</tr>
		<tr>
			<td>Blocking solution 10x</td>
			<td>Telo TAGGG Telomere Length Assay kit</td>
			<td>12209136001</td>
			<td></td>
		</tr>
		<tr>
			<td>Cutsmart Buffer</td>
			<td>NEB</td>
			<td>B6004</td>
			<td></td>
		</tr>
		<tr>
			<td>Detection buffer 10x</td>
			<td>Telo TAGGG Telomere Length Assay kit</td>
			<td>12209136001</td>
			<td></td>
		</tr>
		<tr>
			<td>Dig easy hyb</td>
			<td>Telo TAGGG Telomere Length Assay kit</td>
			<td>12209136001</td>
			<td></td>
		</tr>
		<tr>
			<td>Digestion Buffer</td>
			<td>Telo TAGGG Telomere Length Assay kit</td>
			<td>12209136001</td>
			<td></td>
		</tr>
		<tr>
			<td>Hinf 1</td>
			<td>Telo TAGGG Telomere Length Assay kit</td>
			<td>12209136001</td>
			<td></td>
		</tr>
		<tr>
			<td>Hinf 1 (alternative to kit)</td>
			<td>NEB</td>
			<td>R0155T</td>
			<td></td>
		</tr>
		<tr>
			<td>Loading Dye</td>
			<td>BIOLABS</td>
			<td>N3231S</td>
			<td></td>
		</tr>
		<tr>
			<td>Maleic acid buffer 10x</td>
			<td>Telo TAGGG Telomere Length Assay kit</td>
			<td>12209136001</td>
			<td></td>
		</tr>
		<tr>
			<td>Molecular marker</td>
			<td>Telo TAGGG Telomere Length Assay kit</td>
			<td>12209136001</td>
			<td></td>
		</tr>
		<tr>
			<td>Probe</td>
			<td>Telo TAGGG Telomere Length Assay kit</td>
			<td>12209136001</td>
			<td></td>
		</tr>
		<tr>
			<td>Rsa 1</td>
			<td>Telo TAGGG Telomere Length Assay kit</td>
			<td>12209136001</td>
			<td></td>
		</tr>
		<tr>
			<td>Rsa 1 (alternative to kit)</td>
			<td>NEB</td>
			<td>R0167L</td>
			<td></td>
		</tr>
		<tr>
			<td>Substrate</td>
			<td>Telo TAGGG Telomere Length Assay kit</td>
			<td>12209136001</td>
			<td></td>
		</tr>
		<tr>
			<td>Wash buffer</td>
			<td>Telo TAGGG Telomere Length Assay kit</td>
			<td>12209136001</td>
			<td></td>
		</tr>
	</tbody>
</table>

optimization of performance parameters of the taggg telomere length assay

端粒是存在于染色体末端的重复序列;它们的缩短是人类体细胞的特征。缩短是由于末端复制的问题和端粒酶的缺失而发生的，端粒酶负责维持端粒长度。有趣的是，端粒也会响应各种内部生理过程而缩短，如氧化应激和炎症，这些过程可能由于污染物、传染因子、营养物质或辐射等细胞外因素而受到影响。因此，端粒长度是衰老和各种生理健康参数的极好生物标志物。TAGGG 端粒长度测定试剂盒用于使用端粒限制性片段 （TRF） 测定来量化平均端粒长度，并且具有高度可重复性。然而，这是一种昂贵的方法，因此，它不常规用于大样本数量。在这里，我们描述了一个详细的方案，用于使用南方印迹或TRF分析以及基于非放射性化学发光的检测来优化且经济高效地测量端粒长度。

Telomeres are the repetitive DNA sequences present at the end of chromosomes. They have tandem repeats of TTAGGG and maintain genome integrity by protecting the chromosome from both fraying and the end replication problem, which means that part of the 3&#39; overhang is unable to be replicated by DNA polymerase1,2. Short telomeres lead to chromosomal abnormalities in cells, due to which cells become permanently arrested in a stage called replicative senescence3. Short telomeres also cause a host of other problems, such as mitochondria dysfunction4,5 and cell dysfunction.
DNA telomeric repeats are lost as and when the cell divides, with an average loss of 25 to 200 bp per year6, resulting in cellular senescence after a certain number of divisions6. Aging is associated with a higher frequency of comorbidities, which is marked by a shortening in telomere length7. Telomere restriction fragment (TRF) analysis, as described by Mender, is a very expensive method8. Because of this, it is not implemented while quantifying telomere length in most studies.
Presently, the majority of epidemiological studies employ quantitative polymerase chain reaction (qPCR)-based measurements of telomere length. However, the qPCR-based method is a relative measurement method, as it measures the ratio between telomeres and single-copy gene amplification products, and not absolute telomere length. Telomere length measurement using the TRF protocol is the gold standard method, as it can measure telomere length distribution in the sample and measurements can be expressed in absolute values in kilobases (kb). However, its use is limited because it is cumbersome, labor-intensive, and costly. Here, we present an optimized protocol for telomere length measurement using chemiluminescence-based TRFs.
TRF analysis includes seven major steps: 1) culturing of cells for genomic DNA extraction, 2) genomic DNA extraction using the phenol:chloroform:isoamylalcohol (P:C:I) method, 3) restriction digestion of genomic DNA, 4) agarose gel electrophoresis, 5) Southern blotting of the restriction digestion DNA fragment, 6) hybridization and detection via chemiluminescence-the immobilized telomere probe is visualized by a highly sensitive chemiluminescent substrate for alkaline phosphatase, disodium 2-chloro-5-(4-methoxyspiro[1,2-dioxetane-3,2&#8242;-(5-chlorotricyclo[3.3.1.13.7]decan])-4-yl]-1-phenyl phosphate (CDP-Star)-and 7) analysis for obtaining mean telomere length and range information from these telomeric smears.

作者聚焦：TAGGG 端粒长度测定性能参数的优化  

TAGGG端粒长度测定性能参数优化

The scope of our research involves measuring telomere length differences across various study groups. The questions we are trying to answer revolve around diseases of physiological conditions and how they may affect telomere length and vice versa. The technology is currently used to advance research in the field of telomere length biology are nanopore sequencing and more recently CRISPR Cas based measurement.Current experimental challenges are accurately measuring absolute telomere length and the changes in it. The same sample could give varying results when different methods of measuring telomere length are used. The research gap we address is the lack of a standard method to measure telomere length.Telomere restriction fragment analysis or TRF, is still the standard against which all new techniques are evaluated. Also, our optimized protocols helps in processing a higher number of samples with limited resources. Our findings will help advance research in the field of telomere length biologic by providing a reliable and relatively economical way to compare newer methods to the gold standard, which is TRF.Our laboratory will focus on the effects of small molecule inhibitors on telomere length. We will also study the impact that lifestyle diseases and telomere length have on on each other.

在1%琼脂糖凝胶上运行的提取的基因组DNA（gDNA）显示出良好的完整性，如图 1B所示，表明该样品可用于TRF的进一步下游处理。然后通过修改每个步骤所需的溶液体积来进行TRF测定（见表 1 和 表2）。TRF 信号清晰可见（图 3）。因此，通过修改溶液体积和浓度，可以处理更多的样品而不会对结果产生任何负面影响，并且可以使用?...

Watch this Scientific Journal Video about Optimization of Performance Parameters of the TAGGG Telomere Length Assay at JoVE.com

在这里，我们详细描述了使用非放射性化学发光检测量化端粒长度的方案，重点是优化TAGGG端粒长度测定试剂盒的各种性能参数，例如缓冲液量和探针浓度。

Telomeres are repetitive sequences which are present at chromosomal ends; their shortening is a characteristic feature of human somatic cells. Shortening ...

我们的研究范围涉及测量不同研究组的端粒长度差异。我们试图回答的问题围绕着生理疾病的疾病以及它们如何影响端粒长度，反之亦然。该技术目前用于推进端粒长度生物学领域的研究，包括纳米孔测序和最近基于CRISPR Cas的测量。目前的实验挑战是准确测量绝对端粒长度及其变化。当使用不同的方法来测量端粒长度时，同一样品可能会产生不同的结果。我们解决的研究差距是缺乏测量端粒长度的标准方法。端粒限制性片段分析（TRF）仍然是评估所有新技术的标准。此外，我们优化的实验方案有助于以有限的资源处理更多的样品。我们的研究结果将有助于推进端粒长度生物学领域的研究，提供一种可靠且相对经济的方法来将新方法与黄金标准（TRF）进行比较。我们的实验室将专注于小分子抑制剂对端粒长度的影响。我们还将研究生活方式疾病和端粒长度对彼此的影响。

optimization-performance-parameters-taggg-telomere-length

Research

JoVE Journal

Cancer Research

Optimization of Performance Parameters of the TAGGG Telomere Length Assay

1.5K 次观看.  SVKM's NMIMS Deemed-to-be-University. 我们的研究范围涉及测量不同研究组的端粒长度差异。我们试图回答的问题围绕着生理疾病的疾病以及它们如何影响端粒长度，反之亦然。该技术目前用于推进端粒长度生物学领域的研究，包括纳米孔测序和最近基于CRISPR Cas的测量。目前的实验挑战是准确测量绝对端粒长度及其变化。当使用不同的方法来测量端粒长度时，同一样品可能会产生不同的结果。我们解决的研...

视频: 作者聚焦：TAGGG 端粒长度测定性能参数的优化  

Telomeres are repetitive sequences which are present at chromosomal ends; their shortening is a characteristic feature of human somatic cells. Shortening occurs due to a problem with end replication and the absence of the telomerase enzyme, which is responsible for maintaining telomere length. Interestingly, telomeres also shorten in response to various internal physiological processes, like oxidative stress and inflammation, which may be impacted due to extracellular agents like pollutants, infectious agents, nutrients, or radiation. Thus, telomere length serves as an excellent biomarker of aging and various physiological health parameters. The TAGGG telomere length assay kit is used to quantify average telomere lengths using the telomere restriction fragment (TRF) assay and is highly reproducible. However, it is an expensive method, and because of this, it is not employed routinely for large sample numbers. Here, we describe a detailed protocol for an optimized and cost-effective measurement of telomere length using Southern blots or TRF analysis and non-radioactive chemiluminescence-based detection.

Here, we describe in detail the protocol for quantifying telomere length using non-radioactive chemiluminescent detection, with a focus on the optimization of various performance parameters of the TAGGG telomere length assay kit, such as buffer quantities and probe concentrations.

https://cloudfront.jove.com/CDNSource/teasers/65288.jpg

科研

癌症研究

A2780 Cells Genomic DNA Isolation and Analysis

Begin by adding 500 microliters of the lysis buffer to the previously obtained A2780 cell pellet, and mix gently using a cut tip with a minimum opening diameter of two millimeters. Add 20 micrograms per milliliter of freshly prepared RNase and mix by gentle inversion. Incubate it at 37 degrees Celsius for 30 minutes.At the end of incubation, add proteinase K to a final concentration of 100 micrograms per milliliter and gently invert 10 times. Incubate again at 55 degrees Celsius for one hour inverting the tube every 10 minutes. Pipette 500 microliters of phenol:chloroform:isoamyl alcohol reagent into the tube and gently invert the tube about 20 times.Centrifuge it at 9, 391 G for 15 minutes at room temperature. Pipette the upper aqueous viscous layer into a new tube. Add an equal volume of chloroform and mix by gentle inversion.After centrifuging the tube once, collect the aqueous layer. Add an appropriate amount of five molar sodium chloride in the tube to increase the concentration by 0.2 moles. Add two volumes of 100%ethanol into the tube and invert it gently 20 times.Centrifuge at 15, 871 G for five minutes at room temperature. After discarding the supernatant, add 500 microliters of 70%ethanol. Centrifuge again at the same condition and then discard the supernatant.Once the pellet is air dried, add 50 microliters of sterile nuclease free water and let it rehydrate. Then mix the DNA by pipetting using the cut tip. Use UV spectrophotometry to measure the concentration of DNA.After digesting the DNA, separate it using 0.8%agarose. Submerge the agarose gel in 0.25 molar hydrochloric acid solution for 10 minutes at room temperature with gentle agitation. Then rinse the gel twice with distilled water.To denature the DNA, submerge the gel in a solution of sodium hydroxide and sodium chloride. Then rinse the gel twice with distilled water. To neutralize the DNA as demonstrated, submerge the gel in a solution of 0.5 molar hydrochloric acid and three molar sodium chloride at pH seven.The extractor genomic DNA showed good integrity indicating its use for further downstream processing of telomere restriction fragments.

A2780 细胞基因组 DNA 分离和分析

Southern Blotting, Hybridization, and Chemiluminescence Detection of Genomic DNA Isolated from A2780 Cells

To perform southern blotting, start by cutting a nylon membrane into a 10 by 12 centimeter sized segment. Make a notch in the same position as the gel. Activate the membrane by dipping it in distilled water, followed by 20X sodium saline citrate buffer.Place a filter paper wick on a double inverted tray, set such that the ends touch the base of the outer tray. Place the gel over the filter paper. Then place the activated nylon membrane over the gel, ensuring that the notches overlap.Remove any air bubbles with a glass rod. Place a two centimeter heap of 9.5 by 11.5 centimeter sized cellulose filter paper, and another six centimeter heap of regular filter paper. Place a weight atop the setup for equal weight distribution.Fill the outer tray with 20X sodium saline citrate buffer, and leave it overnight for efficient transfer. After southern transfer, fix the transferred DNA on the membrane by UV crosslinking on a UV transilluminator. Wash the membrane twice with 25 milliliters of 2X sodium saline citrate buffer.To perform hybridization, incubate the membrane in 10 milliliters of the pre-warmed pre-hybridization buffer. Gently agitate the membrane manually at frequent intervals. Then incubate the blot in 10 milliliters of hybridization buffer at 42 degrees Celsius for three hours with gentle agitation.Wash the blot twice in 25 milliliters of stringent buffer for 10 minutes at room temperature with gentle agitation. Now wash the blot twice in 25 milliliters of pre-warmed stringent buffer at 50 degrees Celsius for 15 minutes with gentle agitation. Rinse with 15 milliliters of wash buffer for five minutes with gentle agitation at room temperature.Incubate the membrane in 10 milliliters of freshly prepared blocking solution for 30 minutes at room temperature with gentle agitation. Then place it in 10 milliliters of anti-digoxigenin conjugated with an alkaline phosphatase working solution. Wash the blot twice in the wash buffer at room temperature for 15 minutes.Now incubate in 10 milliliters of detection buffer for five minutes at room temperature. After removing the excess buffer, place the membrane on an acetate sheet with the DNA side facing up. Add approximately one to 1.5 milliliters of substrate solution dropwise on the membrane.Immediately place another acetate sheet on it and incubate for five minutes at room temperature. Squeeze out the excess substrate solution before imaging. Using a gel documentation imaging system, collect multiple unsaturated images at different time points for analysis.After southern blotting and hybridization, the telomere restriction fragments were clearly visible, indicated by a smear.