我们承认艾米托马森为她准备这篇稿子帮助和弗雷德里克Skarstedt和Ryan克里斯蒂他们在数字的准备工作。

随着机构审查委员会的批准，进行了搜索的电子实验室的数据库来识别福尔马林固定，石蜡包埋（FFPE）代表巨细胞病毒（CMV）感染的胃肠道病例（GI）的诊断经病理组织学活检（苏木块和伊红（H＆E）和/或免疫组织化学（IHC））。 1，从FFPE胃肠道活检组织组织处理<ol><li>收集FFPE胃肠道活检组织块巨细胞病毒（CMV）定量聚合酶链反应（定量PCR）。这些块被存储在室温下。 </li><li>预标签1.7毫升离心管与块的唯一标识号。 </li><li>锁定切片机手轮。 </li><li>座椅的组织块装入纸盒夹。 </li><li>使用新鲜的，未使用的刀片，对准块在切片机刀角。 </li><li>调整切片机切成10微米厚的切片。 </li><li>解锁日Ë手轮和走向刀片块。 </li><li>切5节块在10微米厚的间隔，使每个切滚进组织的画卷。确保每个滚动包含所需的活检组织有足够的代表性。 </li><li>锁定切片机手轮。 </li><li>用钳子，将所有5滚动组织到预标记的离心管中，小心仅由石蜡操纵滚动，以减少潜在的交叉污染。被固定在管帽，并把它放回架上。 </li><li>从切片取出组织块。 </li><li>取下并扔掉刀片。 </li><li>净化可能已经走在与以往组织接触的任何表面或仪器。 </li><li>将一个新鲜的，未使用的刀片插入刀架。 </li><li>对于要处理的每个额外的块重复步骤1.4-1.14。可以存储包含组织滚动离心管在室温下直到添ËDNA提取。 </li></ol>从卷轴2。提取DNA FFPE胃肠道活检组织中注:此协议是改编自FFPE组织DNA提取试剂盒（注意:请参阅材料安全数据表（MSDS））的包装说明书。 <ol><li>加入1 ml二甲苯（注意:请参照MSDS），每个样品的离心管。盖上盖子，涡旋剧烈，持续10秒。 </li><li>继续进行DNA提取在制造商的产品插页中具体概述的进行了以下修改。 </li><li>先加6μL内部控制（IC）的每个离心管中。 <ol><li>继ATL /蛋白酶K裂解液孵育温度56℃，1小时，孵育99℃30分钟，而不是90℃，1小时。 </li><li>在最后的DNA洗脱步骤中，小心地打开DNA纯化柱的盖和应用50μl的蒸馏水DNA酶/ RNA酶的水在膜的中心，而不是buffeřATE。 注:请参考说明书的相应的存储和准备试剂。应的DNA提取所有5 FFPE滚动。执行所有离心步骤在室温（15-25℃）。 </li></ol></li></ol> 3，定量PCR巨细胞病毒<ol><li>聚合酶链反应（PCR）程序<ol><li>生成一个Excel工作表中的PCR为巨细胞病毒检测运行。这个Excel工作表计算所需弥补主结构与镁每种试剂的总量。它乘以每个样品所需的每种试剂的用量（如下所列）样品和对照的次数加一（以补偿损失，由于移液）。 <ol><li>包括在量每个PCR反应如下试剂所示: 试剂0;每个样品量 巨细胞病毒LC预混液小瓶12.5微升 镁溶液黄色小瓶2.5微升 总成交量15.0微升</li></ol></li><li>包括在每一个实验中，运行水或无靶对照（NTC），阴性对照组，低阳性对照，高阳性对照，要么QS3或QS4，所有患者样本。毛细管应设置在此顺序。 </li><li>得到冷却块具体针对被存储在4℃的实时PCR仪器置于适当的细管中的冷却块。请勿触摸毛细管的表面。处理的毛细血管时，请务必使用手套。 </li><li>设置所有聚合酶链反应在清洁油烟机不沾放大生产TS。 <ol><li>从PCR试剂盒中移除主小瓶的正确数目，以测试所有患者样品，加5控制。让主人小瓶，镁溶液，PCR级水解冻的冷却块。每个主管含有足够的试剂进行12项测试。 </li><li>轻轻涡旋主小瓶，并在最大速度快速旋转它们在离心机。 </li><li>结合每个主小瓶的内容到一个小瓶中，并再次轻轻旋涡​​。 </li><li>结合起来，在工作表中的PCR表示成一个单一的无菌离心管的金额母液和Mg的解决方案。 </li><li>轻轻混匀，并等分15μl反应混合液用Mg到每个毛细管，除了在这对定量标准（QS）中第5位的毛细管。 </li></ol></li><li>移动包含毛细管和主结构与镁从清洁油烟机的油烟机加工冷却块。 </li><li>加入2μl内部控制（IC）的其余30μl反应的拌上镁。轻轻混匀后分装15微升至毛细管中的位置5（位置为QS）。 </li><li>移液管加入10μl水，对照DNA或样品DNA插入适当的毛细管。帽用封端工具的毛细管。 </li><li>采取冷却块/样品的实时PCR仪。 </li></ol></li><li>程序的实时PCR仪（ 见表1） </li><li>扩增和检测的实时PCR仪进行<ol><li>登录到计算机</li><li>双击仪器图标，然后登录到软件。 </li><li>打开的实时PCR仪器</li><li>点击从正面屏幕"巨细胞病毒/ EB病毒"。 </li><li>按照宏观向导的提示进行操作。 </li><li>选择框运行自检时，软件提示。自检必须进行每天一次的仪器在使用中。 </li><li>命名运行。 </li><li>调整位于上部来氟米特样本计数包含在运行页面吨角落的对照和患者的总和，并输入每个患者样本的识别号码。 </li><li>点击"绝对定量"选项卡上。 </li><li>根据"样品类型"，指定为标准适当的样品类型。 </li><li>在为定量标准（QS）的预期浓度的类型。 </li><li>从冷却器块取出毛细管并将其放置在转盘中的相应位置（冷却块样品＃1应放置在1号位置上的旋转木马等 ）。 </li><li>将转盘到仪器的离心机，并按下启动。 </li><li>打开离心机除去转盘。 </li><li>放置在传送带进入实时PCR仪。合上盖子。 </li><li>按"开始→运行"。 </li><li>实时PCR仪会检查每个位置的样本。不要走开从仪器上，直到这个检查已经完成，尤其cially如果毛细管的一个不是在传送带上。该仪器会注意到这一点，并询问是否运行将继续进行。临走回答是。 </li><li>一旦运行完成后，关闭生成的报告。 </li><li>点击"绝对定量"。 </li><li>点击旁边的箭头颜色补偿。 </li><li>从下拉框中点击"选择颜色补偿"。 </li><li>选择最新的色彩补偿文件适用于运行。 </li><li>单击OK（确定）时，框弹出。 </li><li>点击箭头由标准曲线。 </li><li>选择"使用外部"，从下拉菜单中。 </li><li>选择最当前的外部标准曲线文件，然后单击确定。 </li><li>再看下的绝对定量菜单中每条曲线以保证曲线的存在，而不是平线，如果浓度分配。的绝对定量为705/Back 530自动分析。 </li></ol></li><li>曲先进而精湛的控制<ol><li>产生每当收到试剂的新批号一个新的标准曲线，或每半年一次，取其更频繁。 </li><li>加入10微升每个标准对母液与镁。考虑包含在套件中先前提取的DNA的标准。 </li><li>加入1μl内部控制的每个标准的主混合物用Mg用于产生标准曲线，以保证准确的定量。不要添加IC到主结构与镁的无模板对照（NTC）。 </li><li>准备一个NTC和定量的标准QS 4，3，2，1，和1:10稀释QS4的运行，以产生标准曲线3次重复，共16个数据点。 </li><li>选择标准重复"重复"中的下拉菜单</li><li>选择通道530/back没有和分析时，运行打开颜色补偿， </li><li>在"标准曲线（在运行）"，选择"另存为前TERNAL"并命名该曲线。保存时输入注释"为标准曲线应用"。 </li><li>格拉夫结果。由此产生的线性相关系数必须≥0.81。 </li><li>日常运行，包括QS3或QS4的一个副本在运行控制。在分析阶段当前外部标准曲线可导入运行。 </li><li>运行控制:无DNA对照，阴性对照，低，高的阳性对照。预计病毒载量是确定和调整各厂商和批号。 </li><li>加的IC到每个样品的DNA提取过程中控制。 </li><li>如果任何控件都属于超出范围，记录不能接受的结果，并参考化验排除故障。 </li></ol></li><li>的频率控制<ol><li>包括在每次运行:一个NTC，阴性，弱阳性，强阳性，和标准的控制。 </li></ol></li></ol>注:可接受的限度控制。无峰应遵守d表示无模板（NTC）和阴性对照。峰值应该出现在530路和预期的病毒载量被确定并调整每个厂商和批号为低和高阳性对照。

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	<li>Hodinka, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Manual of Clnical Microbiology. 2, 1558-1574 (2011).</li><li>Lasry, S., 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=Interstrain+variations+in+the+cytomegalovirus+(CMV)+glycoprotein+B+gene+sequence+among+CMV-infected+children+attending+six+day+care+centers.">Interstrain variations in the cytomegalovirus (CMV) glycoprotein B gene sequence among CMV-infected children attending six day care centers.</a> The Journal of infectious diseases. 174, 606-609 (1996).</li><li>Murph, J. R., Baron, J. C., Brown, C. K., Ebelhack, C. L., Bale Jr, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+occupational+risk+of+cytomegalovirus+infection+among+day-care+providers.">The occupational risk of cytomegalovirus infection among day-care providers.</a> JAMA : the journal of the American Medical Association. 265, 603-608 (1991).</li><li>Pass, R. F., 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=Vaccine+prevention+of+maternal+cytomegalovirus+infection.">Vaccine prevention of maternal cytomegalovirus infection.</a> The New England journal of medicine. 360, 1191-1199 (2009).</li><li>McCoy, M. 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=qPCR+increases+sensitivity+to+detect+cytomegalovirus+in+paraffin-embedded,+formalin-fixed+tissue+of+gastrointestinal+biopsies.">qPCR increases sensitivity to detect cytomegalovirus in paraffin-embedded, formalin-fixed tissue of gastrointestinal biopsies.</a> Human Pathology. 45, 48-53 (2014).</li><li>Mills, A. M., Guo, F. P., Copland, A. P., Pai, R. K., Pinsky, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparison+of+CMV+detection+in+gastrointestinal+mucosal+biopsies+using+immunohistochemistry+and+PCR+performed+on+formalin-fixed,+paraffin-embedded+tissue.">A comparison of CMV detection in gastrointestinal mucosal biopsies using immunohistochemistry and PCR performed on formalin-fixed, paraffin-embedded tissue.</a> The American journal of surgical pathology. 37, 995-1000 (2013).</li><li>Krech, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complement-fixing+antibodies+against+cytomegalovirus+in+different+parts+of+the+world.">Complement-fixing antibodies against cytomegalovirus in different parts of the world.</a> Bulletin of the World Health Organization. 49, 103-106 (1973).</li><li>Chou, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Newer+methods+for+diagnosis+of+cytomegalovirus+infection.">Newer methods for diagnosis of cytomegalovirus infection.</a> Reviews of infectious diseases. 12 Suppl 7, (1990).</li><li>Brytting, M., Xu, W., Wahren, B., Sundqvist, V. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytomegalovirus+DNA+detection+in+sera+from+patients+with+active+cytomegalovirus+infections.">Cytomegalovirus DNA detection in sera from patients with active cytomegalovirus infections.</a> Journal of clinical microbiology. 30, 1937-1941 (1992).</li><li>Revello, M. G., 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=Human+cytomegalovirus+in+blood+of+immunocompetent+persons+during+primary+infection:+prognostic+implications+for+pregnancy.">Human cytomegalovirus in blood of immunocompetent persons during primary infection: prognostic implications for pregnancy.</a> The Journal of infectious diseases. 177, 1170-1175 (1998).</li><li>Shinkai, M., Bozzette, S. A., Powderly, W., Frame, P., Spector, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Utility+of+urine+and+leukocyte+cultures+and+plasma+DNA+polymerase+chain+reaction+for+identification+of+AIDS+patients+at+risk+for+developing+human+cytomegalovirus+disease.">Utility of urine and leukocyte cultures and plasma DNA polymerase chain reaction for identification of AIDS patients at risk for developing human cytomegalovirus disease.</a> The Journal of infectious diseases. 175, 302-308 (1997).</li></ol>

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许多实验室方法可用于巨细胞病毒的诊断病毒（CMV）感染，包括血清学，病毒培养，分子血症测定法，活组织检查材料的组织学，并且，最近表明，分子的福尔马林固定，石蜡包埋（FFPE）的活检测定组织5,6。血清学检查，一旦确诊的中流砥柱，只有可靠地识别风险，并不会与急性CMV感染7很好的相关性。病毒培养，常规和早期抗原壳小瓶中，通过长时间的测定时间的阻碍，在后者<...

的巨细胞病毒（CMV）感染在临床标本中的解释是非常重要的。巨细胞病毒是疱疹病毒的betaherpesvirinae亚家族的成员。类似于其他疱疹病毒，巨细胞病毒必须建立持续性或隐性感染者的能力特征是缺乏病毒复制活跃1。在压力或其他免疫抑制的时间可能会发生病毒的再活化，导致病毒复制活跃其特征在于，病毒体释放，这可以伴随疾病表现2-4。胃肠道（GI），巨细胞病毒病的症状通常包括腹部疼痛和/或血便1。 巨细胞病毒性肠胃炎的组织学诊断采用苏木精伊红（H＆E），以确定巨细胞病毒包涵体。在临床上怀疑是高还没有明显的包裹体观察病例，免疫组织化学（IHC）经常作为一种辅助的测试方法。然而，IHC也可以通过稀有非经典出现细胞染色模式（模棱两可）的阻碍，使得难以解释（ 图2）5。它试图利用DNA从福尔马林固定，石蜡包埋（FFPE）的GI活检组织和定量聚合酶链反应（定量PCR）来检测CMV胃肠活检萃取。该技术已被证明是有价值的巨细胞病毒在胃肠活检的检测，并且特别有用的模棱两可的IHC染色例5,6。此外，定量PCR也显示出额外的巨细胞病毒临床试验数据以及相关可用时6。在检测巨细胞病毒使用的qPCR可能会导致早期诊断和治疗的情况下，临床怀疑为巨细胞病毒是很高，但H＆E和CMV免疫组化是否定的。

<table><tbody><tr><td>1.7 ml Microfuge tubes</td><td>Costar</td><td>3620</td><td></td></tr><tr><td>Serrated forceps</td><td>Electron Microscopy Services</td><td>62086-1S</td><td>Micro Forceps MF-1</td></tr><tr><td>Tabletop centrifuges (microfuge)</td><td>Eppendorf</td><td>5424</td><td></td></tr><tr><td>Xylene</td><td>Fisher</td><td>Fisher Scientific: X3P</td><td>1 gallon</td></tr><tr><td>Ethanol</td><td>Fisher</td><td>Fisher Scientific: ET108</td><td>96-100%</td></tr><tr><td>Microtome</td><td>Leica</td><td>RM2255</td><td></td></tr><tr><td>QIAamp DNA FFPE Tissue Kit</td><td>Qiagen</td><td>56404</td><td></td></tr><tr><td>artus CMV LightCycler PCR ASR</td><td>Qiagen</td><td>4500025</td><td></td></tr><tr><td>CMV LC PCR Supplement Kit</td><td>Qiagen</td><td>1031873</td><td></td></tr><tr><td>LightCycler centrifuge</td><td>Roche</td><td>75005087</td><td></td></tr><tr><td>LightCycler Cooling Block</td><td>Roche</td><td>10800058001</td><td></td></tr><tr><td>LightCycler Capping Tool</td><td>Roche</td><td>3357317</td><td></td></tr><tr><td>Color Compensation Kit</td><td>Roche</td><td>2158850</td><td></td></tr><tr><td>LightCycler 20 &amp;mu;l Capillaries</td><td>Roche</td><td>1 909 339</td><td></td></tr><tr><td>Blades</td><td>Sakura</td><td>Fisher Scientific: 4689</td><td>Accu-Edge low profile</td></tr></tbody></table>

qpcr is a sensitive and rapid method for detection of cytomegaloviral dna in formalin-fixed, paraffin-embedded biopsy tissue

关键的是要确定在胃肠道（GI）的免疫抑制病人的呼吸道巨细胞病毒（CMV）感染，给出了发展为严重感染的风险更大。已经开发了许多实验室方法为巨细胞病毒感染的检测，包括血清学，病毒培养和分子生物学方法。通常情况下，这些方法反映全身受累的巨细胞病毒，不具体确定局部组织的参与。因此，在胃肠道中检测CMV感染是通过活检组织的传统组织学频繁进行。苏木精伊红（H＆E）染色与免疫组织化学（IHC）结合仍然检查这些切片的支柱。 H＆E和免疫组化有时会导致非典型（模棱两可）染色模式，使解释困难。它表明，定量聚合酶链反应（定量PCR）对CMV可以成功地对福尔马林固定，石蜡包埋（FFPE）的活检组织为非常执行高敏感性和特异性。本协议的目的是演示如何在临床实验室的设置进行定量PCR检测巨细胞病毒在FFPE组织活检的检测。这种方法很可能是为病人大有裨益的胃肠活检模棱两可染色巨细胞病毒的病例。

共有228组织块用定量聚合酶链反应（定量PCR），这是由91巨细胞病毒（CMV）阳性例可疑CMV免疫组化的基础上组织学和积极的免疫组织化学（IHC），18，79和阴性对照试验。 如图2，巨细胞病毒阳性病例会表现出典型的巨细胞病毒包涵体（A）和/或阳性IHC染色（B）。模棱两可的情况下会表现出罕见的，非经典的出现染色模式（C），而阴性对照会显示?...

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qPCR Is a Sensitive and Rapid Method for Detection of Cytomegaloviral DNA in Formalin-fixed, Paraffin-embedded Biopsy Tissue

This protocol describes qPCR detection of cytomegalovirus in formalin-fixed, paraffin-embedded biopsy tissue, which is rapid, sensitive, specific, and useful for interpreting equivocal hematoxylin and eosin or immunohistochemical staining patterns.

定量PCR是用于检测巨细胞病毒DNA在福尔马林固定，石蜡包埋的活检组织一个灵敏快速的方法

It is crucial to identify cytomegalovirus (CMV) infection in the gastrointestinal (GI) tract of immunosuppressed patients, given their greater risk for ...

qpcr-is-sensitive-rapid-method-for-detection-cytomegaloviral-dna

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

15.7K 次观看.  Indiana University School of Medicine. This protocol describes qPCR detection of cytomegalovirus in formalin-fixed, paraffin-embedded biopsy tissue, which is rapid, sensitive, specific, and useful for interpreting equivocal hematoxylin and eosin or immunohistochemical staining patterns.

视频: qPCR Is a Sensitive and Rapid Method for Detection of Cytomegaloviral DNA in Formalin-fixed, Paraffin-embedded Biopsy Tissue

Protocol for HER2 FISH Using a Non-cross-linking, Formalin-free Tissue Fixative to Combine Advantages of Cryo-preservation and Formalin Fixation

Morphologic assessment of formalin-fixed, paraffin-embedded (FFPE) tissue samples has been the gold standard for cancer diagnostics for decades due to its excellent preservation of morphology. Personalized medicine increasingly provides individually adapted and targeted therapies for characterized individual diseases enabled by combined morphological and molecular analytical technologies and diagnostics. Performance of morphologic and molecular assays from the same FFPE specimen is challenging because of the negative impact of formalin due to chemical modification and cross-linking of nucleic acids and proteins. A non-cross-linking, formalin-free tissue fixative has been recently developed to fulfil both requirements, i.e., to preserve morphology like FFPE and biomolecules like cryo-preservation. Since FISH is often required in combination with histopathology and molecular diagnostics, we tested the applicability of FISH protocols on tissues treated with this new fixative. We found that formalin post-fixation of histological sections of non-cross-linking, formalin-free and paraffin-embedded (NCFPE) breast cancer tissue generated equivalent results to those with FFPE tissue in human epidermal growth factor receptor 2 (HER2) FISH analysis. This protocol describes how a FISH assay originally developed and validated for FFPE tissue can be used for NCFPE tissues by a simple post-fixation step of histological sections.

Fluorescence in-situ hybridization (FISH) is often required in combination with histopathology and molecular diagnostics for selection of therapy in personalized medicine. A novel non-cross-linking, formalin-free tissue fixative that allows high quality morphologic, molecular and FISH analyses from the same specimen by addition of a post-fixation step before FISH is presented.

非交联无甲醛组织固定剂 HER2 鱼的协议, 结合低温保存和福尔马林固色的优点

Morphologic assessment of formalin-fixed, paraffin-embedded (FFPE) tissue samples has been the gold standard for cancer diagnostics for decades due to its ...

科研

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. ...

Detection of a CDH1 Rare Transcript Variant in Fresh-frozen Gastric Cancer Tissues by Chip-based Digital PCR

CDH1a, a non-canonical transcript of the CDH1 gene, has been found to be expressed in some gastric cancer (GC) cell lines, whereas it is absent in normal gastric mucosa. Recently, we detected CDH1a transcript variant in fresh-frozen tumor tissues obtained from patients with GC. The expression of this variant in tissue samples was investigated by the chip-based digital PCR (dPCR) approach presented here. dPCR offers the potential for an accurate, robust, and highly sensitive measurement of nucleic acids and is increasingly utilized for many applications in different fields. dPCR is capable of detecting rare targets; in addition, dPCR offers the possibility for absolute and precise quantification of nucleic acids without the need for calibrators and standard curves. In fact, the reaction partitioning enriches the target from the background, which improves amplification efficiency and tolerance to inhibitors. Such characteristics make dPCR an optimal tool for the detection of the CDH1a rare transcript.

Detection of a CDH1 Rare Transcript Variant in Fresh-frozen Gastric Cancer Tissues by Chip-based Digital PCR

The manuscript describes a chip-based digital PCR assay to detect a rare CDH1 transcript variant (CDH1a) in fresh-frozen normal and tumor tissues obtained from patients with gastric cancer.

基于芯片的数字 PCR 检测新鲜冷冻胃癌组织中的一个CDH1罕见转录变体

CDH1a, a non-canonical transcript of the CDH1 gene, has been found to be expressed in some gastric cancer (GC) cell lines, whereas it is absent in normal ...

Retrospective MicroRNA Sequencing: Complementary DNA Library Preparation Protocol Using Formalin-fixed Paraffin-embedded RNA Specimens

&#8211;Archived, clinically classified formalin-fixed paraffin-embedded (FFPE) tissues can provide nucleic acids for retrospective molecular studies of cancer development. By using non-invasive or pre-malignant lesions from patients who later develop invasive disease, gene expression analyses may help identify early molecular alterations that predispose to cancer risk. It has been well described that nucleic acids recovered from FFPE tissues have undergone severe physical damage and chemical modifications, which make their analysis difficult and generally requires adapted assays. MicroRNAs (miRNAs), however, which represent a small class of RNA molecules spanning only up to ~18&#8211;24 nucleotides, have been shown to withstand long-term storage and have been successfully analyzed in FFPE samples. Here we present a 3&#39; barcoded complementary DNA (cDNA) library preparation protocol specifically optimized for the analysis of small RNAs extracted from archived tissues, which was recently demonstrated to be robust and highly reproducible when using archived clinical specimens stored for up to 35 years. This library preparation is well adapted to the multiplex analysis of compromised/degraded material where RNA samples (up to 18) are ligated with individual 3&#39; barcoded adapters and then pooled together for subsequent enzymatic and biochemical preparations prior to analysis. All purifications are performed by polyacrylamide gel electrophoresis (PAGE), which allows size-specific selections and enrichments of barcoded small RNA species. This cDNA library preparation is well adapted to minute RNA inputs, as a pilot polymerase chain reaction (PCR) allows determination of a specific amplification cycle to produce optimal amounts of material for next-generation sequencing (NGS). This approach was optimized for the use of degraded FFPE RNA from specimens archived for up to 35 years and provides highly reproducible NGS data.

Formalin-fixed paraffin-embedded specimens represent a valuable source of molecular biomarkers of human diseases. Here we present a laboratory-based cDNA library preparation protocol, initially designed with fresh frozen RNA, and optimized for the analysis of archived microRNAs from tissues stored up to 35 years.

回顾性 MicroRNA 排序: 用福尔马林固定石蜡嵌入 RNA 标本补充 DNA 文库制备协议

&#8211;Archived, clinically classified formalin-fixed paraffin-embedded (FFPE) tissues can provide nucleic acids for retrospective molecular studies of ...

遗传学

High Sensitivity 5-hydroxymethylcytosine Detection in Balb/C Brain Tissue

DNA hydroxymethylation is a long known modification of DNA, but has recently become a focus in epigenetic research. Mammalian DNA is enzymatically modified at the 5th carbon position of cytosine (C) residues to 5-mC, predominately in the context of CpG dinucleotides. 5-mC is amenable to enzymatic oxidation to 5-hmC by the Tet family of enzymes, which are believed to be involved in development and disease. Currently, the biological role of 5-hmC is not fully understood, but is generating a lot of interest due to its potential as a biomarker. This is due to several groundbreaking studies identifying 5-hydroxymethylcytosine in mouse embryonic stem (ES) and neuronal cells. Research techniques, including bisulfite sequencing methods, are unable to easily distinguish between 5-mC and 5-hmC . A few protocols exist that can measure global amounts of 5-hydroxymethylcytosine in the genome, including liquid chromatography coupled with mass spectrometry analysis or thin layer chromatography of single nucleosides digested from genomic DNA. Antibodies that target 5-hydroxymethylcytosine also exist, which can be used for dot blot analysis, immunofluorescence, or precipitation of hydroxymethylated DNA, but these antibodies do not have single base resolution.In addition, resolution depends on the size of the immunoprecipitated DNA and for microarray experiments, depends on probe design. Since it is unknown exactly where 5-hydroxymethylcytosine exists in the genome or its role in epigenetic regulation, new techniques are required that can identify locus specific hydroxymethylation. The EpiMark 5-hmC and 5-mC Analysis Kit provides a solution for distinguishing between these two modifications at specific loci. The EpiMark 5-hmC and 5-mC Analysis Kit is a simple and robust method for the identification and quantitation of 5-methylcytosine and 5-hydroxymethylcytosine within a specific DNA locus. This enzymatic approach utilizes the differential methylation sensitivity of the isoschizomers MspI and HpaII in a simple 3-step protocol. Genomic DNA of interest is treated with T4-BGT, adding a glucose moeity to 5-hydroxymethylcytosine. This reaction is sequence-independent, therefore all 5-hmC will be glucosylated; unmodified or 5-mC containing DNA will not be affected. This glucosylation is then followed by restriction endonuclease digestion. MspI and HpaII recognize the same sequence (CCGG) but are sensitive to different methylation states. HpaII cleaves only a completely unmodified site: any modification (5-mC, 5-hmC or 5-ghmC) at either cytosine blocks cleavage. MspI recognizes and cleaves 5-mC and 5-hmC, but not 5-ghmC. The third part of the protocol is interrogation of the locus by PCR. As little as 20 ng of input DNA can be used. Amplification of the experimental (glucosylated and digested) and control (mock glucosylated and digested) target DNA with primers flanking a CCGG site of interest (100-200 bp) is performed. If the CpG site contains 5-hydroxymethylcytosine, a band is detected after glucosylation and digestion, but not in the non-glucosylated control reaction. Real time PCR will give an approximation of how much hydroxymethylcytosine is in this particular site. In this experiment, we will analyze the 5-hydroxymethylcytosine amount in a mouse Babl/C brain sample by end point PCR.

The EpiMark 5-hmC and 5-mC Analysis Kit can be used to analyze and quantitate 5-methylcytosine and 5-hydroxymethylcytosine within a spe cific locus. The kit distinguishes 5-mC from 5-hmC by the addition of glucose to the hydroxyl group of 5-hmC via an enzymatic reaction utilizing &beta;-glucosyltransferase (T4-BGT). When 5-hmC occurs In the context of CCGG, this modification converts a cleavable MspI site to a non-cleavable site.

BALB / C脑组织中的高灵敏度检测5 - hydroxymethylcytosine

DNA hydroxymethylation is a long known modification of DNA, but has recently become a focus in epigenetic research.   Mammalian DNA is enzymatically ...

神经科学

Detection and Monitoring of Tumor Associated Circulating DNA in Patient Biofluids

Complications associated with upfront and repeat surgical tissue sampling present the need for minimally invasive platforms capable of molecular sub-classification and temporal monitoring of tumor response to therapy. Here, we describe our dPCR-based method for the detection of tumor somatic mutations in cell free DNA (cfDNA), readily available in&#160;patient biofluids. Although limited in the number of mutations that can be tested for in each assay, this method provides a high level of sensitivity and specificity. Monitoring of mutation abundance, as calculated by MAF, allows for the evaluation of tumor response to therapy, thereby providing a much-needed supplement to radiographic imaging.

Here, we present a protocol to detect tumor somatic mutations in circulating DNA present in patient biological fluids (biofluids). Our droplet digital polymerase chain reaction (dPCR)-based method enables quantification of the tumor mutation allelic frequency (MAF), facilitating a minimally invasive complement to diagnosis and temporal monitoring of tumor response.

患者生物流体中肿瘤相关循环DNA的检测与监测

Complications associated with upfront and repeat surgical tissue sampling present the need for minimally invasive platforms capable of molecular ...

Detection of the Genome and Transcripts of a Persistent DNA Virus in Neuronal Tissues by Fluorescent In situ Hybridization Combined with Immunostaining

Single cell codetection of a gene, its RNA product and cellular regulatory proteins is critical to study gene expression regulation. This is a challenge in the field of virology; in particular for nuclear-replicating persistent DNA viruses that involve animal models for their study. Herpes simplex virus type 1 (HSV-1) establishes a life-long latent infection in peripheral neurons. Latent virus serves as reservoir, from which it reactivates and induces a new herpetic episode. The cell biology of HSV-1 latency remains poorly understood, in part due to the lack of methods to detect HSV-1 genomes in situ in animal models. We describe a DNA-fluorescent in situ hybridization (FISH) approach efficiently detecting low-copy viral genomes within sections of neuronal tissues from infected animal models. The method relies on heat-based antigen unmasking, and directly labeled home-made DNA probes, or commercially available probes. We developed a triple staining approach, combining DNA-FISH with RNA-FISH&#160;and immunofluorescence, using peroxidase based signal amplification to accommodate each staining requirement. A major improvement is the ability to obtain, within 10 &#181;m tissue sections, low-background signals that can be imaged at high resolution by confocal microscopy and wide-field conventional epifluorescence. Additionally, the triple staining worked with a wide range of antibodies directed against cellular and viral proteins. The complete protocol takes 2.5 days to accommodate antibody and probe penetration within the tissue.

Detection of the Genome and Transcripts of a Persistent DNA Virus in Neuronal Tissues by Fluorescent In situ Hybridization Combined with Immunostaining

We established a fluorescent in situ hybridization protocol for the detection of a persistent DNA virus genome within tissue sections of animal models. This protocol enables studying infection process by codetection of the viral genome, its RNA products, and viral or cellular proteins within single cells.

检测持久性病毒的DNA在神经组织​​中的荧光的基因组与成绩单原位杂交结合免疫组化染色

Single cell codetection of a gene, its RNA product and cellular regulatory proteins is critical to study gene expression regulation. This is a challenge ...

In Situ Detection of Bacteria within Paraffin-embedded Tissues Using a Digoxin-labeled DNA Probe Targeting 16S rRNA

The presence of bacteria within the pocket epithelium and underlying connective tissue in gingival biopsies from patients with periodontitis has been reported using various methods, including electron microscopy, immunohistochemistry or immunofluorescence using bacteria-specific antibodies, and fluorescent in situ hybridization (FISH) using a fluorescence-labeled oligonucleotide probe. Nevertheless, these methods are not widely used due to technical limitation or difficulties. Here a method to localize bacteria within paraffin-embedded tissues using DIG-labeled DNA probes has been introduced. The paraffin-embedded tissues are the most common form of biopsy tissues available from pathology banks. Bacteria can be detected either in a species-specific or universal manner. Bacterial signals are detected as either discrete forms (coccus, rod, fusiform, and hairy form) of bacteria or dispersed forms. The technique allows other histological information to be obtained: the epithelia, connective tissue, inflammatory infiltrates, and blood vessels are well distinguished. This method can be used to study the role of bacteria in various diseases, such as periodontitis, cancers, and inflammatory immune diseases.

In Situ Detection of Bacteria within Paraffin-embedded Tissues Using a Digoxin-labeled DNA Probe Targeting 16S rRNA

Here a method to localize bacteria within paraffin-embedded tissues using DIG-labeled 16S rRNA-targeting DNA probes has been described. This protocol can be applied to study the role of bacteria in various diseases such as periodontitis, cancers, and inflammatory immune diseases.

原位检测使用地高辛标记的DNA探针石蜡包埋组织中的细菌靶向16S rRNA基因

The presence of bacteria within the pocket epithelium and underlying connective tissue in gingival biopsies from patients with periodontitis has been ...

免疫与感染

增强癌症组织样本中致癌驱动因素的 FISH 分析

Robust Detection of Gene Amplification in Formalin-Fixed Paraffin-Embedded Samples by Fluorescence In Situ Hybridization

Focal gene amplification, such as extrachromosomal DNA (ecDNA), plays an important role in cancer development and therapy resistance. While sequencing-based methodologies enable an unbiased identification of ecDNA, cytogenetic-based techniques, such as fluorescence in situ hybridization (FISH), remain time and cost-effective for identifying ecDNA in clinical specimens. The application of FISH in formalin-fixed paraffin-embedded (FFPE) tissue samples offers a unique avenue for detecting amplified genes, particularly when viable specimens are not available for karyotype examination. However, there is a lack of consensus procedures for this technique. This protocol provides comprehensive, fully optimized, step-by-step instructions for conducting FISH to detect gene amplification, including ecDNA, in FFPE tissue samples which present unique challenges that this protocol aims to overcome and standardize. By following this protocol, researchers can reproducibly acquire high-quality imaging data to assess gene amplification.

作者聚焦：FISH 作为癌症标本中精确基因扩增评估的工具

This protocol provides a reproducible method to visualize gene amplification in formalin-fixed paraffin-embedded (FFPE) tissue specimens.

定量PCR是用于检测巨细胞病毒DNA在福尔马林固定，石蜡包埋的活检组织一个灵敏快速的方法

摘要

探索更多视频

此视频中的章节

非交联无甲醛组织固定剂 HER2 鱼的协议, 结合低温保存和福尔马林固色的优点

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

基于芯片的数字 PCR 检测新鲜冷冻胃癌组织中的一个CDH1罕见转录变体

回顾性 MicroRNA 排序: 用福尔马林固定石蜡嵌入 RNA 标本补充 DNA 文库制备协议

BALB / C脑组织中的高灵敏度检测5 - hydroxymethylcytosine

患者生物流体中肿瘤相关循环DNA的检测与监测

检测持久性病毒的DNA在神经组织中的荧光的基因组与成绩单原位杂交结合免疫组化染色

原位检测使用地高辛标记的DNA探针石蜡包埋组织中的细菌靶向16S rRNA基因

通过荧光原位杂交对福尔马林固定石蜡包埋样品中的基因扩增进行稳健检测

通过荧光原位杂交对福尔马林固定石蜡包埋样品中的基因扩增进行稳健检测

定量PCR是用于检测巨细胞病毒DNA在福尔马林固定，石蜡包埋的活检组织一个灵敏快速的方法

摘要

探索更多视频

此视频中的章节

非交联无甲醛组织固定剂 HER2 鱼的协议, 结合低温保存和福尔马林固色的优点

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

基于芯片的数字 PCR 检测新鲜冷冻胃癌组织中的一个CDH1罕见转录变体

回顾性 MicroRNA 排序: 用福尔马林固定石蜡嵌入 RNA 标本补充 DNA 文库制备协议

BALB / C脑组织中的高灵敏度检测5 - hydroxymethylcytosine

患者生物流体中肿瘤相关循环DNA的检测与监测

检测持久性病毒的DNA在神经组织​​中的荧光的基因组与成绩单原位杂交结合免疫组化染色

原位检测使用地高辛标记的DNA探针石蜡包埋组织中的细菌靶向16S rRNA基因

通过荧光原位杂交对福尔马林固定石蜡包埋样品中的基因扩增进行稳健检测

通过荧光原位杂交对福尔马林固定石蜡包埋样品中的基因扩增进行稳健检测

检测持久性病毒的DNA在神经组织中的荧光的基因组与成绩单原位杂交结合免疫组化染色