Name: modified terminal restriction fragment analysis for quantifying telomere length using in-gel hybridization
Uploaded: 2017-07-10T10:30:00.000+00:00
Duration: 11 min 29 s
Description: Watch this Scientific Journal Video about Modified Terminal Restriction Fragment Analysis for Quantifying Telomere Length Using In-gel Hybridization at JoVE.com

作者承认匹兹堡大学癌症研究所生物行为肿瘤学共享设施的支持，部分由P30CA047904颁发。

基因组DNA提取<ol><li>根据制造商的说明书，使用商业DNA提取试剂盒，从细胞（这里是细胞系BJ-hTERT）中进行基因组DNA提取。 </li><li>用分光光度计测定DNA浓度。如果DNA浓度小于100μg/ mL，则用乙醇沉淀DNA，并重悬于一定体积的TE缓冲液（10mM Tris-Cl; 0.1mM EDTA（乙二胺四乙酸）; pH8.0）中），以确保DNA浓度100-600μg/ mL）。 </li></ol> DNA完整性评估注意:本协议部分概述了使用11 cm x 14 cm凝胶凝胶电泳系统的DNA完整性评估。也可以使用其他系统和凝胶尺寸，但电压和DNA迁移时间可能会有所不同。 <ol><li>通过将1g电泳级琼脂糖与100ml TAE缓冲液（40mM Tr是基; 20mM乙酸; 2mM EDTA; pH 8.5）和微波直到琼脂糖溶解并且溶液澄清。在室温下冷却溶液至达到50℃（约15-20分钟）。 </li><li>当琼脂糖溶液冷却时，通过向铸造托盘添加铸造端浇口来制备凝胶铸造托盘。为了防止琼脂糖泄漏，请在连接到铸造托盘之前将端盖冷却至4°C。 </li><li>一旦琼脂糖溶液冷却至50°C，加入10μL溴化乙锭（10mg / mL，在dH 2 O中），并将琼脂糖溶液倒入凝胶浇铸盘中。将梳子加入铸造托盘。 </li><li>让琼脂糖溶液在室温下硬化45分钟。 </li><li>通过使用1x TE缓冲液稀释25ng基因组DNA至终体积为9μL并加入1μL10x载色染料（0.25％（w / v）溴酚蓝，0.25％（w / v）二甲苯）来准备DNA样品进行电泳cyanol FF，30％（v / v）甘油）。混合好</li><li&gt;通过用1x TAE缓冲液填充电泳系统准备凝胶电泳系统进行DNA迁移，使缓冲液水平高于凝胶数毫米。加载凝胶的孔与整个DNA样品。将5μLDNA梯子加入单个孔。 </li><li>在100V下运行DNA迁移约2小时，或直到溴酚蓝染料大约通过凝胶的一半。 </li><li>使用紫外线透照器或等效物对凝胶进行成像。 注意:具有良好完整性的DNA样品具有高分子量带，无涂片（即由于剪切或破碎的DNA）。如果DNA条带被涂抹，则它们具有低完整性，并且不适于Southern印迹分析，并且DNA必须用新鲜细胞重新分离。 </li></ol> 3.基因组DNA消化<ol><li> 以20-40μL的最终体积进行基因组DNA的消化。 注意:容量更大超过40μL会溢出琼脂糖凝胶的孔。 <ol><li>在微管中合并3μg基因组DNA和适量的10x DNA消化缓冲液（限制性内切酶供应），使终浓度为1倍。向每个管中加入1μLRsaI限制酶（10,000U / mL）和1μLHinfI限制酶（10,000U / mL）。使用无菌的去离子H 2 O调节最终体积。充分混合。短暂离心（10 - 15秒，16,000 xg），以确保所有组件都在管的底部。 </li></ol></li><li>在37℃下孵育≥16小时。消化后，如果需要，将消化的DNA在4℃或-20℃下储存不超过2天。 </li></ol> 4.基因组DNA凝胶电泳注意:本协议部分概述了使用20 cm x 25 cm凝胶电泳系统的程序。不同凝胶电可以使用电泳系统，但是可能需要修改几个变量，包括电压，迁移时间和添加到系统中的电泳缓冲液量，以获得最佳结果。 <ol><li>通过将2.25g凝胶电泳级琼脂糖与375mL电泳缓冲液，1×TAE或0.5×TBE（110mM Tris碱; 90mM硼酸; 2.5mM EDTA; pH8.3）组合来制备0.6％琼脂糖溶液。 注意:对于小于8,000 kB的端粒，建议使用0.5x TBE，而推荐使用1×TAE作为8,000 kB至20,000 kB 1之间的端粒。 </li><li>微波溶液，直到琼脂糖溶解，溶液澄清。让溶液在室温下冷却15-20分钟，或直至琼脂糖达到50℃。 </li><li>当琼脂糖溶液冷却时，如步骤2.2所述，制备凝胶电泳系统进行凝胶注射。将琼脂糖凝胶溶液倒入凝胶浇铸盘中。把梳子放进去凝胶。让凝胶固化45分钟，露在室温下。 </li><li>准备消化的DNA样品进行电泳。对于40 uL反应，向每个消化的DNA样品中加入10μL负载染料0.4μL。简单地离心（10-15秒，16,000 xg），以确保所有的溶液都在管的底部。 </li><li>准备DNA迁移的凝胶电泳系统。从凝胶上取下末端门和梳子。将填充线的1x TAE或0.5x TBE的凝胶电泳系统填充，确保凝胶完全浸没在缓冲液中。 </li><li>用DNA样品加载凝胶孔，使样品之间有一个空白孔。避免引起气泡或刺穿凝胶。将10μLDNA梯子加入凝胶两端的孔中。 注意:DNA梯子的类型将取决于端粒的长度，这将随人与人之间和细胞来源而异。 </li><li>运行凝胶电泳150 V 30 min，快速将DNA转移到凝胶中;然后将电压调整至57 V，运行18-24 h。 </li></ol>凝胶荧光成像和杂交<ol><li> 18-24小时后，关闭凝胶电泳电源。用凝胶从电泳缓冲液中取出凝胶浇注托盘。从凝胶的右上角切片，作为凝胶取向的参考。 </li><li>通过将纸张切割成比凝胶稍大的尺寸来准备一张滤纸。将滤纸放置在铸造托盘中的凝胶顶部，并使纸张吸收凝胶上的多余溶液。使用移液器作为滚筒小心地去除滤纸和凝胶之间的气泡，以通过侧面挤压气泡。 </li><li>通过翻转凝胶和滤纸，使纸张位于凝胶下方，从铸造托盘中取出凝胶。将凝胶用滤纸转移到凝胶干燥器上并覆盖凝胶用塑料包装。使用移液器作为滚筒，去除塑料包装和凝胶之间的所有气泡。 </li><li>在50℃下将凝胶干燥2小时。 </li><li>一旦干燥，取出塑料包装，并将凝胶和滤纸转移到含有250mL去离子水（足以覆盖凝胶）的玻璃皿上。仔细取出滤纸，并在室温下孵育凝胶15分钟。凝胶会变薄，坚固，易于操作而不会撕裂。 </li><li>在去离子水中制备5x SSC溶液（750 mM NaCl; 75 mM柠檬酸钠）。 </li><li>将凝胶放在尼龙网（12英寸×12英寸）的顶部。将尼龙网和凝胶卷在一起，确保凝胶不与其自身接触。将网眼和凝胶放入杂交管（ 例如 ，直径为1.5的管中为12）。 注意:正确卷起时，凝胶将与两侧的尼龙网接触。 </li><li>结合100 mL 5x SSC和12μL绿色荧光核酸acid染色，并放入杂交管。保护溶液免受光照，并在旋转的杂交炉中在42℃孵育30分钟。 </li><li>从杂交管中取出尼龙网/凝胶，滚动打开，放入含有250毫升去离子水的玻璃皿中，轻轻取出尼龙网。使用磷光体图像凝胶。使用荧光设置:520 BP，40蓝色和488nm。将光电倍增管（PMT）设置为375，焦平面为+3 mm，灵敏度为正常值。将图像保存到文件。 </li></ol>杂交<ol><li> 准备放射性端粒探针。 <ol><li>合并2μL（2 ng）端粒探针（5-（CCC TAA） 4 3&#39;），2.5μL10x多核苷酸激酶反应缓冲液，3μLγ32 P-dATP（6,000 Ci / mmol），4μLT4多核苷酸激酶和无DNA酶，无菌去离子水，最终体积为20μL离心管 注意:使用放射性时应遵守设施的放射性安全和法规。 </li></ol></li><li>简单离心（10-30秒，16,000 xg），并在37℃的水浴中孵育1小时。以16,000 xg离心10-30秒，并在65℃下孵育20分钟以停止反应。 </li><li>使用G-25色谱微柱，通过涡旋混合色谱柱的内容物，然后在室温下以700×g离心1分钟。丢弃滤液。 </li><li>将放射性探针溶液装入G-25色谱柱，并以700 x g离心2分钟。保存滤液。 注意:保留滤液，因为它含有放射性端粒探针。如果需要，放射性探针可以在4°C储存。 </li><li>在无菌去离子水中制备250 mL由1.5 M NaCl和0.5 M NaOH组成的变性溶液。 </li><li>将凝胶放入含有250 mL变性溶液和培养基的玻璃皿中在室温下食用15分钟。取出变性溶液，并用250毫升无菌去离子水代替。在室温下在轨道振荡器（40-50rpm）上孵育10分钟。 </li><li>在无菌去离子水中制备250 mL由1.5 NaCl和0.5 M Tris-盐酸组成的中和溶液，pH 8.0。 </li><li>取出去离子水，并用250 mL中和溶液代替，并在室温下孵育15分钟。 </li><li>在中和溶液培养过程中，制备50μlSSC，5×Denhardt溶液（0.1％非离子合成聚合物; 0.1％聚乙烯吡咯烷; 0.1％牛血清白蛋白），10mM磷酸二钠（Na 2 HPO 4 ）和1mM焦磷酸钠四水合物（Na 4 P 2 O 7· 10H 2 O）的无水去离子水中。 </li><li>将凝胶卷成尼龙网，放入杂交管（步骤5.7）。加入20 mL杂交溶液，并在42°C的杂交烘箱中旋转孵育10 min。 </li><li>取出杂交溶液，并用20 mL新鲜杂交溶液代替。加入整个端粒探针，并在42℃的杂交烘箱中旋转过夜。 </li></ol>凝胶洗涤，荧光屏曝光和无线电成像<ol><li>在无菌去离子水中制备500 mL 2x SSC溶液（300 mM NaCl; 30 mM柠檬酸钠）。 </li><li>在去离子水中准备250 mL 0.1x SSC（15 mM NaCl; 1.5 mM柠檬酸钠）和0.1％十二烷基硫酸钠（SDS）。 </li><li>从杂交管中取出凝胶并筛网，并放入含250 mL 2x SSC的玻璃皿中。展开并从盘中取出尼龙网。将玻璃皿放在轨道摇床上，室温孵育15分钟。 </li><li>取出2x SSC，并用250 mL 0.1x SSC和0代替.1％SDS溶液，并在室温下在轨道振荡器上孵育15分钟。 </li><li>孵化期间，将磷光体屏幕曝光至图像橡皮擦15分钟。 </li><li>取出0.1x SSC和0.1％SDS溶液，并用250 mL 2x SSC取代。在轨道摇床上孵育15分钟。 </li><li>从2×SSC溶液中取出凝胶，并将凝胶包裹在塑料包装中或放入可热封袋中。清除凝胶和塑料包装或塑料袋之间的所有气泡和气泡。使用Phopshor屏幕将凝胶放入曝光盒。将荧光屏暴露于凝胶过夜。 </li><li>使用磷光体图像曝光的荧光屏。 </li></ol> 8.端粒长度定量<ol><li>打开包含绿色荧光核酸染色凝胶的荧光图像的磷光体文件（ 图1 ）。 </li><li>选择"工具"，然后选择"像素距离"9 ;.使用鼠标，从凝胶底部绘制一条线到第一个标记带的中间并记录距离。对每个标记带重复此过程（ 图2 ）。这些距离将用于图形程序中，如下所述。 </li><li>打开放射性标记凝胶的磷光体图 （ 图3 ）。选择"对象管理器"，然后选择"网格"。将网格设置为1列和150行。调整网格，使其从凝胶顶部延伸到凝胶底部。调整网格的宽度等于车道的宽度，点击网格边缘，拖动网格的宽度等于车道的宽度。 </li><li>复制此网格（以使框的宽度和高度保持不变），并将第二个网格移动到空的通道（为了背景目的）。继续复制和移动其他样品车道的附加网格。 注意:完成后，应该有每个样品通道的网格和一个空格的背景网格（ 图4 ）。 </li><li>选择"分析"，然后选择"卷报告"，选择报告的所有网格，显示卷报告后，双击报告激活电子表格程序，保存电子表格文件，该文件的数据将为在"卷"车道下。 </li><li>打开一个合适的图形程序并创建一个新的数据和表。选择"Y:输入并绘制每个点的单个Y值"，然后选择"创建"。 </li><li>将第一列（列X）标记为"测量距离"，并将第二列（列Y）标记为"分子量"。输入列Y中每个标记带的标记分子量。输入对应于每个分子量标记的步骤8.2中获得的迁移距离。 </li><li>进入"分析"，选择"分析"，然后选择"非线性回归"on（曲线拟合）&#39;，然后选择"OK"。在下一个屏幕上，选择"指数"，然后选择"单相衰减"。选择"范围"选项卡，并在"定义曲线的点数"中输入"150"，然后选中"创建XY坐标表以导出或将曲线复制到另一个程序"。 注意:结果"数据1的非线性拟合"包含Y行中每个距离的分子量。 </li><li>对于样品通道的分析，选择高于和低于实际端粒带的分析区域。不要使用车道的整个长度（ 图5 ）。确定每个车道分析区域的网格位置。 </li><li>选择"文件"，"新建"创建新的表格和图表。选择"Y:输入并绘制每个点的单个Y值"，然后选择"创建"。从电子表格文件复制并粘贴背景通道卷值（强度值）进入第一列（X）和从样品通道到第一列Y的体积值（强度值）。如果有多个样品通道，则将每一组体积值从电子表格文件复制到图形程序中的其他Y列文件。 </li><li>删除在步骤8.8中确定的分析区域之外的后台列（X）和每个样品通道（Y）的值。 注意:数据表仅应包含与分析区域对应的网格位置中的值。 </li><li> 选择分析，然后选择"分析"和"转换"。点击"确定"。选择"Y = YX变换Y值"。这将转换数据2（变换强度值（Y）=样本强度（Y） - 背景强度（X））中的数据。 <ol><li>选择分析，然后选择"分析"，"列分析"和"列统计"。选择确定。结果标签&#39;Col.数据2&#39;的变化数据显示在"Sum"行下，Σ（Int i ）。 </li></ol></li><li> 选择"文件"，"新建"，"创建新表格和图形"。选择"Y:输入并绘制每个点的单个Y值"，然后选择"创建"。将Y列数据从结果页面"Nonline fit Data 1，Curve"复制并粘贴到新的X列中。从结果页面"数据2的转换"复制并粘贴Y列数据到新的Y列。选择分析然后"分析"和"变换"。点击"确定"。选择"使用Y = Y / X转换Y值"。 <ol><li>选择分析，然后选择"分析"，"列分析"和"列统计"。选择确定。结果标签&#39;Col.数据3&#39;的变换数据显示在"Sum"行下，Σ（Int i / MW i ）。 MW =分子量。 </li></ol></li><li>要计算每个样品的平均端粒长度（TL），请使用公式TL =Σ（Int i ）/Σ（Int i / MW i ）。 </li></ol>

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Telomere Shortening as a Marker of Cellular Senescence.</a> Aging (Albany NY). 8 (1), 3-11 (2016).</li><li>Opresko, P. L., Shay, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Telomere-Associated+Aging+Disorders.">Telomere-Associated Aging Disorders.</a> Ageing Res Rev. , 1568-1637 (2016).</li><li>Chiodi, I., Mondello, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Telomere+and+Telomerase+Stability+in+Human+Diseases+and+Cancer.">Telomere and Telomerase Stability in Human Diseases and Cancer.</a> Front Biosci (Landmark Ed). 1 (21), 203-224 (2016).</li><li>Blackburn, E. H., Epel, E. S., Lin, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+Telomere+Biology:+A+Contributory+and+Interactive+Factor+in+Aging,+Disease+Risks,+and+Protection.">Human Telomere Biology: A Contributory and Interactive Factor in Aging, Disease Risks, and Protection.</a> Science. 350 (6265), 1193-1198 (2015).</li><li>Lindqvist, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Psychiatric+Disorders+and+Leukocyte+Telomere+Length:+Underlying+Mechanisms+Linking+Mental+Illness+with+Cellular+Aging.">Psychiatric Disorders and Leukocyte Telomere Length: Underlying Mechanisms Linking Mental Illness with Cellular Aging.</a> Neurosci Biobehav Rev. 55, 333-364 (2015).</li><li>Bodelon, C., Savage, S. A., Gadalla, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Telomeres+in+Molecular+Epidemiology+Studies.">Telomeres in Molecular Epidemiology Studies.</a> Prog Mol Biol Transl Sci. 125 (113), (2014).</li><li>Kimura, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+telomere+length+by+the+Southern+blot+analysis+of+terminal+restriction+fragment+lengths.">Measurement of telomere length by the Southern blot analysis of terminal restriction fragment lengths.</a> Nat Protoc. 5 (9), 1596-1607 (2010).</li><li>Haussmann, M. F., Mauck, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TECHNICAL+ADVANCES:+New+strategies+for+telomere-based+age+estimation.">TECHNICAL ADVANCES: New strategies for telomere-based age estimation.</a> Mol Ecol Resour. 8 (2), 264-274 (2008).</li><li>Nussey, D. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+telomere+length+and+telomere+dynamics+in+evolutionary+biology+and+ecology.">Measuring telomere length and telomere dynamics in evolutionary biology and ecology.</a> Methods Ecol Evol. 5 (4), 299-310 (2014).</li><li>Delany, M. E., Krupkin, A. B., Miller, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organization+of+telomere+sequences+in+birds:+evidence+for+arrays+of+extreme+length+and+for+in+vivo+shortening.">Organization of telomere sequences in birds: evidence for arrays of extreme length and for in vivo shortening.</a> Cytogenet Cell Genet. 90 (1-2), 139-145 (2000).</li><li>Baerlocher, G. M., Vulto, I., de Jong, G., Lansdorp, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometry+and+FISH+to+measure+the+average+length+of+telomeres+(flow+FISH).">Flow cytometry and FISH to measure the average length of telomeres (flow FISH).</a> Nat Protoc. 1 (5), 2365-2376 (2006).</li><li>Sanders, J. L., Newman, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Telomere+length+in+epidemiology:+a+biomarker+of+aging,+age-related+disease,+both,+or+neither.">Telomere length in epidemiology: a biomarker of aging, age-related disease, both, or neither.</a> Epidemiol Rev. 35, 112-131 (2013).</li><li>Parikh, D., Fouquerel, E., Murphy, C. T., Wang, H., Opresko, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Telomeres+are+partly+shielded+from+ultraviolet-induced+damage+and+proficient+for+nucleotide+excision+repair+of+photoproducts.">Telomeres are partly shielded from ultraviolet-induced damage and proficient for nucleotide excision repair of photoproducts.</a> Nat Commun. 6, 8214 (2015).</li></ol>

作者没有什么可以披露的。

电泳后，基因组DNA涂片高于800 bp。如果限制酶有故障或需要其他酶（如上所述），则可能发生这种情况。第二个可能的解释是蛋白质仍然附着在DNA上。当使用分光光度计测定DNA浓度时，260/280的比值应在1.8左右。如果该比例&lt;1.5，则存在可能干扰限制酶消化的蛋白质污染。 DNA样本需要重新分离，否则蛋白质需要用蛋白酶K消化和苯酚:氯仿提取，然后乙醇沉淀除去。商业DNA分离试剂盒的使用常规地?...

位于染色体末端的端粒是在保护细胞遗传信息3,4,5中起关键作用的序列TTAGGG（在人染色体上）的核苷酸重复序列。端粒长度为数千个碱基对，用于在DNA复制过程中保护染色体其余部分的完整性。染色体的复制并不完美，导致染色体的末端未完全复制。这种复制的低效率被称为"终端复制问题"，并导致在每一轮复制6,7期间部分端粒重复的丧失。通过端粒酶细胞分裂后可以恢复端粒长度，端粒酶替代丢失的端粒序列8,9 。然而，端粒酶不是在大多数人体细胞中活化，因此随着时间的流逝，端粒会缩短，最终导致细胞衰老10 。由于端粒缩短，端粒被认为是年龄相关疾病的老化和风险的生物标志物11,12 。此外，端粒长度可能受到遗传，环境和生活方式因素和其他刺激物如氧化应激的影响，表明端粒长度可以作为毒理学，流行病学和行为研究中的生物标志物发挥作用13,14,15 。 本文介绍了使用改性末端限制性片段（TRF）分析法，从Mender和Shay 2以及Kimura 等人测定端粒长度的技术。 16 ，类似于赫伯特，谢伊和里格ht 1和Haussmann和Mauck 17 。传统上，TRF分析涉及Southern印迹法。南方印迹被认为是研究端粒长度的"金标准"，并积极用于检查流行病学研究中的白细胞端粒长度。这种TRF分析使用消化的基因组DNA，留下端粒重复。然后通过凝胶电泳分离DNA片段，变性并转移到硝酸纤维素膜上。端粒序列与端粒寡核苷酸探针杂交。不是将分离的端粒转移到膜上，其他TRF技术使用在DNA变性和不变性的凝胶内杂交技术。当考虑到间质端粒的检测时，包含变性是重要的，这已经在一些物种中报道了19 。缺乏变性步骤的程序只能检测到e末端端单链DNA突出端并不会与间质端粒序列结合18 。 除了TRF分析外，还有其他几种测量端粒长度的技术，每种技术都有自己的优点和缺点。 Flow-FISH技术可以使用流式细胞术16,20测量不同细胞类型的个体细胞的平均端粒长度。虽然它可以用高度特异性的探针精确地标记端粒，并通过自动化来减少人为的错误，但Flow-FISH价格昂贵，效率较低，需要新鲜样品和高技能的技术人员，限制了流行病学研究的能力。此外，该方法不考虑细胞群体染色体数量的潜在变化。 qPCR的另一种技术被广泛接受为测量流行病学研究的端粒长度的手段21 。 TRF程序有其自身的缺陷，限制了其对某些研究的使用。与其他方法（每个样品2至3μg）相比，TRF技术需要大量的DNA。这限制了该技术的应用，以检查可以收集足够的基因组DNA的临床样品的端粒长度，并且不能用于降解的DNA样品。此外，TRF分析费用高昂且劳动密集，需要4-5天才能产生结果。然而，TRF产生低的方差系数授予其重复性。虽然该方案与传统的Southern印迹（利用原位凝胶杂交）不同，但两种技术基本相同。 这里提出的技术是Southern印迹和凝胶内杂交的组合;将Southern印迹的DNA变性步骤与凝胶内杂交结合。与Southern印迹相比，这种组合具有改进的探针信号强度的附加优点，并且在我们的实验室内一直产生可量化的结果。此外，使用放射性探针而不是化学发光探针产生更高的信号强度，并允许使用磷光显像仪进行可视化和定量，从而使TRF用户友好的分析。

<table><tbody><tr><td>&lt;strong&gt;Biologics&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Rsa1, restriction enzyme</td><td>New England Biolabs, Inc</td><td>R0167S</td><td>10,000 U/mL, DNA digestion.&amp;nbsp; Comes with Cutsmart Buffer (digestion buffer)</td></tr><tr><td>Hinf1, restriction enzyme</td><td>New England Biolabs, Inc</td><td>R0155S</td><td>10,000 U/mL, DNA digestion.&amp;nbsp; Comes with Cutsmart Buffer (digestion buffer)</td></tr><tr><td>Seakem GTG Agarose</td><td>Lonza</td><td>50071</td><td>electrophoresis grade agarose</td></tr><tr><td>Optikinase&amp;nbsp;</td><td>affymetrix</td><td>78334X 500 UN</td><td>T4 Polynucleotide Kinase</td></tr><tr><td>SYBR Green Nucleic Acid Stain I</td><td>ThermoFisher Scientific</td><td>S7567</td><td>Syber Green</td></tr><tr><td>exactGene DNA Ladder 24- kB</td><td>Fisher Scientific</td><td>BP2580100</td><td></td></tr><tr><td>C-Strand Probe (3&#039;-(G3AT2)4-&#039;5)</td><td>Integrated DNA Technologies</td><td></td><td>Custom ordered DNA oligonucleotide</td></tr><tr><td>Gamma-ATP-P32</td><td>Perkin Elmer</td><td>BLU502Z</td><td>Radioisotope</td></tr><tr><td>Qiagen Blood and Cell Culture DNA Mini Kit</td><td>Qiagen</td><td>13323</td><td>DNA extraction kit</td></tr><tr><td>GE Illustra Microspin G-25 column</td><td>GE Healthcare Life Sciences</td><td>27-5325-01</td><td>For purification of readioactive oligo probe</td></tr><tr><td>Ficoll 400</td><td></td><td></td><td>non-ionic synthetic polymer</td></tr><tr><td>&lt;strong&gt;Equipment/Software&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Owl A5 Gel electrophoresis system (20 cm x 25 cm)</td><td>ThermoFisher Scientific</td><td>A5</td><td>Gel electrophoresis</td></tr><tr><td>Horizon 11.4 Gel electrophoresis system (11 cm x 14 cm)</td><td>Corel Life Sciences</td><td>11068020</td><td>Gel electrophoresis</td></tr><tr><td>Nylon mesh, 50, 12&quot; x 12&quot;</td><td>Ted Pellam, Inc</td><td>41-12105</td><td></td></tr><tr><td>GE Storage Phosphor Screens</td><td>GE Healthcare Life Sciences</td><td>28-9564-75</td><td></td></tr><tr><td>Phosphor Screen Exposure Cassette</td><td>GE Healthcare Life Sciences</td><td>63-0035-44</td><td></td></tr><tr><td>Isotemp Hybridzation Incubator</td><td>Fisher Scientific</td><td>13-247-20Q</td><td></td></tr><tr><td>Cylindrical hybridization tubes</td><td>Fisher Scientific</td><td>13-247-300</td><td></td></tr><tr><td>The Belly Dancer orbital shaker</td><td>Sigma-aldrich</td><td>Z768499-1EA</td><td>Orbital shaker</td></tr><tr><td>Typhoon 9400</td><td>ABI</td><td></td><td>For imaging of gels and phosphor screens</td></tr><tr><td>GraphPad Prism 6</td><td>GraphPad</td><td>Version 6.05</td><td>Graphing Program</td></tr><tr><td>Microsoft Excel</td><td>Microsoft</td><td>Office 365</td><td>Spreadsheet program</td></tr><tr><td>Nanodrop</td><td>Thermo Scientific</td><td>Nanodrp 2000</td><td>Spectrophotometer</td></tr></tbody></table>

modified terminal restriction fragment analysis for quantifying telomere length using in-gel hybridization

测量端粒长度有几种不同的技术，每种技术都有自己的优点和缺点。传统方法，端粒限制片段（TRF）分析利用DNA杂交技术，其中基因组DNA样品用限制酶消化，留下端粒DNA重复序列和一些亚端粒DNA。将其通过琼脂糖凝胶电泳分离，转移至滤膜并与标记有化学发光或放射性的寡核苷酸探针杂交以观察端粒限制性片段。这种方法虽然需要比其他技术如PCR更大量的DNA，但可以测量细胞群的端粒长度分布，并允许以绝对千碱基表示的测量。该手稿展示了用于确定端粒长度的修饰DNA杂交程序。基因组DNA首先用限制酶消化（不切割）端粒），并通过琼脂糖凝胶电泳分离。然后将凝胶干燥并将DNA变性并原位杂交到放射性标记的寡核苷酸探针。这种原位杂交避免了端粒DNA的损失，并提高信号强度。杂交后，使用荧光屏成像凝胶，并使用图形程序对端粒长度进行定量。该程序由Drs实验室开发。德克萨斯大学西南部1号林德林·赖特和杰瑞·谢伊。在这里，我们将对此过程进行详细说明，并进行一些修改。

分析从细胞系BJ-hTERT（端粒酶永生化人包皮成纤维细胞） 22和人外周血单核细胞（PBMC）分离的三种浓度的DNA（1,2和3μg）端粒长度。 表1显示了每个DNA样品的泳道分配。 图1显示了凝胶的核酸荧光染色。分子量标准清晰可见。确定从凝胶顶部到每个分子量标准的距离（ 图2 ）。 图3</st...

Watch this Scientific Journal Video about Modified Terminal Restriction Fragment Analysis for Quantifying Telomere Length Using In-gel Hybridization at JoVE.com

Modified Terminal Restriction Fragment Analysis for Quantifying Telomere Length Using In-gel Hybridization

在本文中，讨论了使用修饰的末端限制性片段分析量化端粒长度的详细方案，其提供了端粒长度的快速和有效的直接测量。该技术可以应用于DNA的各种细胞来源以量化端粒长度。

使用凝胶内杂交来定量端粒长度的修饰末端限制片段分析

There are several different techniques for measuring telomere length, each with their own advantages and disadvantages. The traditional approach, Telomere ...

modified-terminal-restriction-fragment-analysis-for-quantifying

Research

JoVE Journal

Cancer Research

12.8K 次观看.  University of Pittsburgh. 在本文中，讨论了使用修饰的末端限制性片段分析量化端粒长度的详细方案，其提供了端粒长度的快速和有效的直接测量。该技术可以应用于DNA的各种细胞来源以量化端粒长度。 

视频: Modified Terminal Restriction Fragment Analysis for Quantifying Telomere Length Using In-gel Hybridization

Examination of the Telomere G-overhang Structure in Trypanosoma brucei

The telomere G-overhang structure has been identified in many eukaryotes including yeast, vertebrates, and Trypanosoma brucei. It serves as the substrate for telomerase for de novo telomere DNA synthesis and is therefore important for telomere maintenance. T. brucei is a protozoan parasite that causes sleeping sickness in humans and nagana in cattle. Once infected mammalian host, T. brucei cell regularly switches its surface antigen to evade the host's immune attack. We have recently demonstrated that the T. brucei telomere structure plays an essential role in regulation of surface antigen gene expression, which is critical for T. brucei pathogenesis. However, T. brucei telomere structure has not been extensively studied due to the limitation of methods for analysis of this specialized structure. We have now successfully adopted the native in-gel hybridization and ligation-mediated primer extension methods for examination of the telomere G-overhang structure and an adaptor ligation method for determination of the telomere terminal nucleotide in T. brucei cells. Here, we will describe the protocols in detail and compare their different advantages and limitations.

Examination of the Telomere G-overhang Structure in Trypanosoma brucei

Telomeres are essential for chromosome stability and the telomere G-overhang structure is essential for telomerase-mediated telomere maintenance. We have recently adopted two methods for detecting the telomere G-overhang structure in Trypanosoma brucei, which are native in-gel hybridization and ligation-mediated primer extension, which will be described.

审核端粒G -悬结构锥虫布氏</em

The telomere G-overhang structure has been identified in many eukaryotes including yeast, vertebrates, and Trypanosoma brucei. It serves as the substrate ...

科研

免疫与感染

Laparoscopic Pancreatoduodenectomy With Modified Blumgart Pancreaticojejunostomy

Minimally invasive pancreatic resections are technically demanding but rapidly increasing in popularity. In contrast to laparoscopic distal pancreatectomy, laparoscopic pancreatoduodenectomy (LPD) has not yet obtained wide acceptance, probably due to technical challenges, especially regarding the pancreatic anastomosis.
The study describes and demonstrates all steps of LPD, including the modified Blumgart pancreaticojejunostomy. Indications for LPD are all pancreatic and peri-ampullary tumors without vascular involvement. Relative contra-indications are body mass index &#62;35 kg/m2, chronic pancreatitis, mid-cholangiocarcinomas and large duodenal cancers.
The patient is in French position, 6 trocars are placed, and dissection is performed using an (articulating) sealing device. A modified Blumgart end-to-side pancreaticojejunostomy is performed with 4 large needles (3/0) barbed trans-pancreatic sutures and 4 to 6 duct-to-mucosa sutures using 5/0 absorbable multifilament combined with a 12 cm, 6 or 8 Fr internal stent using 3D laparoscopy. Two surgical drains are placed alongside the pancreaticojejunostomy.
The described technique for LPD including a modified Blumgart pancreatico-jejunostomy is well standardized, and its merits are currently studied in the randomized controlled multicenter trial. This complex operation should be performed at high-volume centers where surgeons have extensive experience in both open pancreatic surgery and advanced laparoscopic gastro-intestinal surgery.

Laparoscopic pancreatoduodenctomy (LPD) may offer advantages over open pancreatoduodenectomy, including early postoperative mobilization, less delayed gastric emptying and a shorter hospital stay. However, LPD is technically challenging and not well-standardized, especially regarding the pancreatic anastomosis. We describe a standardized technique for the pancreatic anastomosis during LPD: modified Blumgart pancreaticojejunostomy.

改良 Blumgart Pancreaticojejunostomy 腹腔镜胰十二指肠切除术

Minimally invasive pancreatic resections are technically demanding but rapidly increasing in popularity. In contrast to laparoscopic distal ...

癌症研究

Simple and Rapid Method to Obtain High-quality Tumor DNA from Clinical-pathological Specimens Using Touch Imprint Cytology

It is critical to determine the mutational status in cancer before administration and treatment of specific molecular targeted drugs for cancer patients. In the clinical setting, formalin-fixed paraffin-embedded (FFPE) tissues are widely used for genetic testing. However, FFPE DNA is generally damaged and fragmented during the fixation process with formalin. Therefore, FFPE DNA is sometimes not adequate for genetic testing because of low quality and quantity of DNA. Here we present a method of touch imprint cytology (TIC) to obtain genomic DNA from cancer cells, which can be observed under a microscope. Cell morphology and cancer cell numbers can be evaluated using TIC specimens. Furthermore, the extraction of genomic DNA from TIC samples can be completed within two days. The total amount and quality of TIC DNA obtained using this method was higher than that of FFPE DNA. This rapid and simple method allows researchers to obtain high-quality DNA for genetic testing (e.g., next generation sequencing analysis, digital PCR, and quantitative real time PCR) and to shorten the turnaround time for reporting results.

Obtaining high-quality genomic DNA from tumor tissues is an essential first step for analyzing genetic alterations using next generation sequencing. In this article, we present a simple and rapid method to enrich tumor cells and obtain intact DNA from touch imprint cytology specimens.

用触印细胞学方法快速获得临床病理标本中高质量肿瘤 DNA

It is critical to determine the mutational status in cancer before administration and treatment of specific molecular targeted drugs for cancer patients. ...

Generation of Cancer Cell Clones to Visualize Telomeric Repeat-containing RNA TERRA Expressed from a Single Telomere in Living Cells

Telomeres are transcribed, giving rise to telomeric repeat-containing long noncoding RNAs (TERRA), which have been proposed to play important roles in telomere biology, including heterochromatin formation and telomere length homeostasis. Recent findings revealed that TERRA molecules also interact with internal chromosomal regions to regulate gene expression in mouse embryonic stem (ES) cells. In line with this evidence, RNA fluorescence in situ hybridization (RNA-FISH) analyses have shown that only a subset of TERRA transcripts localize at chromosome ends. A better understanding of the dynamics of TERRA molecules will help define their function and mechanisms of action. Here, we describe a method to label and visualize single-telomere TERRA transcripts in cancer cells using the MS2-GFP system. To this aim, we present a protocol to generate stable clones, using the AGS human stomach cancer cell line, containing MS2 sequences integrated at a single subtelomere. Transcription of TERRA from the MS2-tagged telomere results in the expression of MS2-tagged TERRA molecules that are visualized by live-cell fluorescence microscopy upon co-expression of a MS2 RNA-binding protein fused to GFP (MS2-GFP). This approach enables researchers to study the dynamics of single-telomere TERRA molecules in cancer cells, and it can be applied to other cell lines.

Here, we present a protocol to generate cancer cell clones containing a MS2 sequence tag at a single subtelomere. This approach, relying on the MS2-GFP system, enables visualization of the endogenous transcripts of telomeric repeat-containing RNA (TERRA) expressed from a single telomere in living cells.

从活细胞中的单端粒表达的癌症细胞克隆的生成, 以可视化含有端粒重复的 rna terra

Telomeres are transcribed, giving rise to telomeric repeat-containing long noncoding RNAs (TERRA), which have been proposed to play important roles in ...

遗传学

Droplet Digital TRAP (ddTRAP): Adaptation of the Telomere Repeat Amplification Protocol to Droplet Digital Polymerase Chain Reaction

The telomere repeat amplification protocol (TRAP) is the most widely used assay to detect telomerase activity within a given a sample. The polymerase chain reaction (PCR)-based method allows for robust measurements of enzyme activity from most cell lysates. The gel-based TRAP with fluorescently labeled primers limits sample throughput, and the ability to detect differences in samples is restricted to two fold or greater changes in enzyme activity. The droplet digital TRAP, ddTRAP, is a highly sensitive approach that has been modified from the traditional TRAP assay, enabling the user to perform a robust analysis on 96 samples per run and obtain absolute quantification of the DNA (telomerase extension products) input within each PCR. Therefore, the newly developed ddTRAP assay overcomes the limitations of the traditional gel-based TRAP assay and provides a more efficient, accurate, and quantitative approach to measuring telomerase activity within laboratory and clinical settings.

Droplet Digital TRAP ddTRAP: Adaptation of the Telomere Repeat Amplification Protocol to Droplet Digital Polymerase Chain Reaction

We successfully converted the standard telomere repeat amplification protocol (TRAP) assay to be employed in droplet digital polymerase chain reactions. This new assay, called ddTRAP, is more sensitive and quantitative, allowing for better detection and statistical analysis of telomerase activity within various human cells.

液滴数字 TRAP (ddTRAP): 对端粒重复放大协议的适应性, 以实现液滴数字聚合酶链反应

The telomere repeat amplification protocol (TRAP) is the most widely used assay to detect telomerase activity within a given a sample. The polymerase ...

<meta charset="utf8"></head><body>人类群体研究中准确的端粒长度评估

Monochrome Multiplex Quantitative PCR Telomere Length Measurement

Telomeres are ribonucleoprotein structures at the end of all eukaryotic chromosomes that protect DNA from damage and preserve chromosome stability. Telomere length (TL) has been associated with various exposures, biological processes, and health outcomes. This article describes the monochrome multiplex quantitative polymerase chain reaction (MMqPCR) assay protocol routinely conducted in our laboratory for measuring relative mean TL from human DNA. There are several different PCR-based TL measurement methods, but the specific protocol for the MMqPCR method presented in this publication is repeatable, efficient, cost-effective, and suitable&#160;for population-based studies. This detailed protocol outlines all information necessary for investigators to establish this assay in their laboratory. In addition, this protocol provides specific steps to increase the reproducibility of TL measurement by this assay, defined by the intraclass correlation coefficient (ICC) across repeated measurements of the same sample. The ICC is a critical factor in evaluating expected power for a specific study population; as such, reporting cohort-specific ICCs for any TL assay is a necessary step to enhance the overall rigor of population-based studies of TL. Example results utilizing DNA samples extracted from peripheral blood mononuclear cells demonstrate the feasibility of generating highly repeatable TL data using this MMqPCR protocol.

<meta charset="utf8"></head><body>作者聚焦：探索创伤对细胞衰老的影响

Here we present a protocol for the measurement of relative telomere length (TL) using the monochrome multiplex quantitative polymerase chain reaction (MMqPCR) assay. The MMqPCR assay is a repeatable, efficient, and cost-effective method for measuring TL from human DNA in population-based studies.

单色多重定量 PCR 端粒长度测量 

Telomeres are ribonucleoprotein structures at the end of all eukaryotic chromosomes that protect DNA from damage and preserve chromosome stability. ...

使用非放射性化学发光检测定量端粒长度

Optimization of Performance Parameters of the TAGGG Telomere Length Assay

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.

作者聚焦：TAGGG 端粒长度测定性能参数的优化  

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.

TAGGG端粒长度测定性能参数优化

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

使用凝胶内杂交来定量端粒长度的修饰末端限制片段分析

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