Name: detection of a cdh1 rare transcript variant in fresh-frozen gastric cancer tissues by chip-based digital pcr
Uploaded: 2018-02-05T15:00:00.000+00:00
Duration: 9 min 16 s
Description: Watch this Scientific Journal Video about Detection of a CDH1 Rare Transcript Variant in Fresh-frozen Gastric Cancer Tissues by Chip-based Digital PCR at JoVE.com

作者希望感谢 Gráinne 蒂尔尼的编辑协助。

该议定书遵循红外人类研究伦理委员会的准则。
注: 本程序专门用于检测人类新鲜冰冻组织中的少量 cDNA 分子。组织切片已被切割在干冰上, 同时仍被冰冻, 从先前经证实的病人衍生的胃肿瘤或正常组织标本。
1. RNA 的分离纯化
<ol><li>
RNA 提取 注意: 使用特定产品在引擎盖下执行 RNA 隔离 (请参见材料表)。但是, 市面上有各种隔离套件。<ol>
<li>质 50-100 毫克的细切新鲜冷冻组织样品在1.5 毫升管与1毫升的 RNA 隔离试剂和漩涡大力十五年代. 在-80 ° c 的夜间孵育管。</li>
		<li>在室温下孵育含有样品的试管5分钟, 并由涡流在十五年代混合。</li>
		<li>保持在冰上的管, 增加200µL 的氯仿, 和漩涡大力十五年代。</li>
		<li>孵育管在室温下为六十年代和离心机在 1.2万 x g 为15分钟在4° c。</li>
		<li>在1.5 毫升管中转移水相, 并加入20µg 的糖原。 注意: 重要的是要小心避免转移任何的界面或有机层, 以减少污染。</li>
		<li>加入500µL 的异丙醇, 倒置管混合, 在冰上孵育10分钟。</li>
		<li>离心管在 1.2万 x g 为15分钟在4° c 沉淀 RNA。 注: RNA 会出现在试管的侧面和底部的凝胶状颗粒中, 通常在离心后是隐形的。</li>
		<li>除去上清液而不干扰沉淀, 用移1毫升75% 乙醇洗涤。</li>
		<li>离心管在 7500 x g 为5分钟在4° c 和去除上清, 不用干扰药丸。</li>
		<li>让颗粒干燥, 直到沉淀变得透明, 并溶解在50µL 的核糖核酸无水。</li>
		<li>在定量之前, 至少在一夜之间将样品冷冻在-80 ° c。</li>
	</ol>
</li>
	<li>
基因组 DNA 清除与 RNA 纯化 注: dnasei 消化步骤, 其次是基于柱的 RNA 纯化, 建议用于分析低丰度目标, 以消化污染的 DNA。<ol>
<li>溶解冻干 dnasei I (1500 Kunitz 单位) 在550µL 的核糖核酸无水使用注射器, 混合轻轻地倒置小瓶, 并将重组的库存解决方案分为等分。</li>
		<li>在一个1.5 毫升的管传输的样本量计算与15µg 的 RNA, 10 µL dnasei 消化缓冲剂 (列于材料表), 2.5 µL dnasei I 库存解决方案, 核糖核酸-免费水到100µL. 室温孵育10分钟。</li>
		<li>添加350µL 的组织裂解缓冲液 (从工具箱中, 请参阅材料表) 并由移混合。</li>
		<li>添加250µL 100% 乙醇和混合移。</li>
		<li>将整个卷 (700 µL) 转换为新的自旋柱, 并在 1.2万 x g 上离心十五年代。</li>
		<li>将过滤器转换为新的收集管, 将500µL 80% 乙醇添加到柱上, 然后在 1.2万 x 克上离心2分钟。</li>
		<li>打开旋转柱的盖子, 在 1.2万 x 克上离心5分钟, 用新的收集管将过滤器烘干;然后将过滤器转到1.5 毫升管。</li>
		<li>将14µL 的无核糖核酸水直接添加到过滤器柱上, 并在 1.2万 x g 离心1分钟。</li>
		<li>保持样品在冰上, 并进行量化使用2µL 的样品在一个台式分光光度计或存储在-80 ° c 的 RNA, 直到使用。</li>
	</ol>
</li>
</ol>2. cDNA 合成
<ol><li>放置 1000 ng RNA, 4 µL 的主混合, 1 µL 的逆转录酶, 和核糖核酸无菌的水到最后的体积20µL 在一个不育的 PCR 管。轻轻地混合, 向下旋转。</li>
	<li>在热循环中孵育反应混合物, 应用以下条件: 5 分钟在25° c, 30 分钟在42° c, 5 min 在85° c。</li>
	<li>离心管的简要, 并进行 dPCR 分析或存储在-20 ° c, 直到需要。</li>
</ol>3. 数字 PCR 反应的建立
<ol><li>
dPCR 反应混合和样品制备
	<ol>
<li>在室温下解冻主混合和化验至少20分钟。</li>
		<li>在6µL 的水中, 将 cDNA 样本稀释到 300 ng 的浓度。</li>
		<li>轻轻地漩涡主混合和准备混合在一个无菌管与8.7 µL 的主混合, 0.87 µL 的CDH1a定制设计的化验底漆, 和1.83 µL 核酸无水的最后量11.4 µL。 注: 对于CDH1a自定义的检测底漆详细信息, 请参阅材料表。</li>
		<li>将准备好的混合物的11.4 µL 转移到稀释的 cDNA 样本, 轻轻地混合, 并简要地离心。 注: 卷包括20% 的过剩, 以弥补移的体积损失。准备所有样品和无模板控制 (NTC) 的混合。</li>
	</ol>
</li>
	<li>
芯片准备 注: 为了获得最佳效果, 请尽快加载芯片。<ol>
<li>插入芯片装载器, 等待指示灯变绿。</li>
		<li>通过轻轻地拉回柱塞 1-2 mm, 卸下浸入式液体注射器的盖子, 并松开它以方便这一步, 并将其替换为提示。</li>
		<li>拿一个新的芯片, 并记下在盖子上写的代码, 将它与样品相关联。</li>
		<li>小心地握住盖子的侧面, 剥去保护膜, 并将盖子与粘脸贴在正确的方向上。</li>
		<li>小心地拿起一块芯片, 小心不要触摸内部部分, 并把它加载到芯片窝在正确的位置, 按下杠杆打开钳。</li>
		<li>加载一个新的加载刀片到加载程序, 并轻轻地推动它, 以确保它是牢固到位。</li>
		<li>将 dPCR 反应混合物的14.5 µL 转移到加载刀片上, 而不制造气泡或偏转刀片, 然后按黑色加载按钮将卷放在芯片上。</li>
		<li>使用浸入式液体注射器转移大约20滴到芯片表面注意不要接触表面与提示。 注意: 重要的是要覆盖整个表面而不溢出液体的边缘。</li>
		<li>旋转装载机臂, 使盖子与芯片接触, 并按下十五年代。</li>
		<li>按下盖子按钮, 释放芯片, 并返回其位置的手臂。</li>
		<li>将组装好的芯片放在45°的角度, 并小心地将浸入式液体与注射器通过填充口, 稍微旋转芯片以确保没有气泡, 并用无菌擦拭去除多余的液体。</li>
		<li>通过轻轻剥离芯片盖顶部的标签, 然后按下填充端口至少5秒来密封芯片机箱。</li>
		<li>将芯片存储在黑暗中, 直到准备好装入热循环。 注: 准备好的芯片应在2小时内使用。</li>
	</ol>
</li>
	<li>
dPCR 反应
	<ol>
<li>打开盖子, 将适配器安装到两个块上, 即使使用了单个块。</li>
		<li>将芯片放在样品块的正确位置。 注: 填充端口必须朝向热循环的前部, 使其处于升高的位置, 以允许任何气泡浮到顶部, 而不会干扰芯片的窗口。用空筹码来平衡两个街区。</li>
		<li>将热垫放在样品块上, 以完全覆盖芯片。</li>
		<li>关闭盖子, 开始 PCR 运行, 应用以下条件: 保持在96° c 10 分钟;45周期60° c 为 2 min 和98° c 为三十年代;保持在60° c 为2分钟;保持在4° c。关闭热循环, 在室温下解冻芯片至少10分钟。 注: 芯片分析必须在一小时内完成。</li>
	</ol>
</li>
	<li>
芯片分析
	<ol>
<li>打开仪表盖, 卸下散热片, 然后从适配器中取出芯片。 注意: 将芯片存放在一个黑暗、干净的位置, 直到分析。</li>
		<li>用异丙醇和无菌擦拭清洗芯片表面。 注意: 检查每个芯片是否有泄漏或潜在的问题。</li>
		<li>将 USB 插入检测器系统以保存数据。</li>
		<li>打开检测系统的芯片托盘, 将芯片正面装入正确位置, 然后关闭托盘。</li>
		<li>等待三十年代进行处理, 然后取出芯片并插入下一条。</li>
		<li>等待分析完成所有处理过的芯片然后插入 USB 到计算机转移文件。 注: 分析的总时间大约是2到3分钟/芯片。</li>
	</ol>
</li>
</ol>4. 数据分析和解释
<ol><li>连接到执行所有下游分析所需的基于云的软件平台。</li>
	<li>创建项目并导入感兴趣的芯片的所有数据文件。</li>
	<li>输入示例名称;选择 "定义芯片" 标签中使用的染料和化验。</li>
	<li>通过在 "审阅数据" 选项卡中进行可视化来确定芯片是否可以接受, 检查该样本在芯片上的加载程度以及评价的数据点。 注: 拒绝少于1.3万评价数据点的芯片。</li>
	<li>移动到屏幕右侧所选芯片的散射图;应用阈值为6000的荧光素 amidite (家) 报告染料信号 (Y 轴) 的所有芯片。 注: 设定的阈值可能根据所使用的化验方法而有所不同。</li>
	<li>通过使用套索工具在散射图上选择相对点, 并按 "待定" 键, 消除任何可疑的正信号以防止误报结果。所有剩余的阳性斑点均表明, 所分析的稀有靶点存在 cDNA 拷贝。</li>
</ol>

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作者报告这项工作没有利益冲突。

dPCR 最初开发了为脱氧核糖核酸分子测量10,11,12,13 , 并且在时间这技术是适应的定量的 rna 并且 RNA 抄本5, 14,15。在本协议中, 我们扩展了应用程序列表, 包括检测来自新鲜冰冻组织样本的稀有记录。为此, 我们使用了相当高的 cDNA (300 ...

CDH1基因对 e-钙黏蛋白进行编码, 这是通过调节细胞黏附、存活、增殖和迁移来维持正常胃上皮的关键因素,1。由于CDH1的有害系或体细胞的改变而导致 e-钙黏附蛋白的丧失, 已与 GC23的开发相关。来自基因内含子2的非规范转录也被假设在胃癌癌变中起到一定作用4,5。特别是, 一个这样的成绩单, CDH1a, 已被证明是用 GC 细胞系表达, 但没有从正常胃4。最近, 我们在 gc 患者使用基于芯片的 dPCR5中检测到气相色谱组织样本中的CDH1a 。dPCR 用于第一次评估在肠道 GC 和正常组织中存在的 CDH1a基因转录。
测定基因表达的金标准方法是实时定量 PCR (qPCR)。但是, 结果数据有时可能是可变的, 质量较差, 尤其是当样本中的目标级别较低时。这种变化可能是由污染物引起的, 它抑制聚合酶活性和底漆退火, 导致非特异的放大和竞争性的副反应6。
虽然 dPCR 的基本生物化学原理与 qPCR 相似, 但 dPCR 显示出一些优势, 允许对基因组 dna (gDNA)/互补 dna (cDNA) 分子进行非常精确的测量。事实上, dPCR 是一个最终点的反应, 它依赖于将一个样本的标定分成数千个井, 因此每个井都包含零个或一个目标分子。放大然后发生在包含目标的拷贝的井, 并且由一个萤光信号指示。然后, 通过用二项式泊松统计值确定正向总分区的比值来计算原始样本中的目标分子的绝对数量7。
此外, dPCR 技术消除了运行标准曲线的需要, 从而避免了相关的偏差和可变性, 从而可以直接量化目标8,9;由于其对抑制剂的高耐受性, 它可以独立于污染物和效率产生更精确和可重现的结果10;它比 qPCR 更灵敏、更特异, 因此是检测稀有靶点的可靠方法。最后, 将样本分成多个反应, 减少了与背景分子的竞争, 提高了目标检测的极限, 使放大成为可能, 并促进了单分子 gDNA/cDNA 的检测6.以芯片为基础的核酸的检测和定量 dPCR 已越来越多地应用于拷贝数变异, DNA 片段的量化, 和突变分析11,12,13, 给出精度和低材料输入要求的方法。另外, dPCR 最近被集成了入分析 rna14和基因抄本5,15。

<table><tbody><tr><td>TRIazol Reagent</td><td>Thermo Fisher Scientific</td><td>15596018</td><td></td></tr><tr><td>Glycogen 20 mg/ml</td><td>ROCHE</td><td>10901393001</td><td></td></tr><tr><td>RNeasy MinElute Cleanup kit</td><td>QIAGEN</td><td>74204</td><td></td></tr><tr><td>iScript cDNA Synthesis kit</td><td>BioRad</td><td>1708891</td><td></td></tr><tr><td>QuantStudio 3D Digital PCR Master Mix v2</td><td>Thermo Fisher Scientific</td><td>A26358</td><td></td></tr><tr><td>CDH1a IDT custom designed assay</td><td>Integrated DNA Technologies (IDT)</td><td>NA</td><td>F) GCTGCAGTTTCACTTTTAGTG&lt;br/&gt; (R) ACTTTGAATCGGGTGTCGAG&lt;br/&gt; (P)/FAM/CGGTCGACAAAGGACAGCCTATT/TAMRA/&lt;br/&gt; [dPCR optimized assay concentrations: 900 nM (F),&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; 900 nM (R), 250 nM (P)]</td></tr><tr><td>QuantStudio 3D Digital PCR 20K Chip Kit v2</td><td>Thermo Fisher Scientific</td><td>A26316</td><td></td></tr><tr><td>Heraeus Biofuge Fresco</td><td>Thermo&amp;nbsp; Scientific</td><td>75002402</td><td></td></tr><tr><td>Thermocycler (Labcycler)</td><td>Sensoquest</td><td>011-103</td><td></td></tr><tr><td>GeneAmp PCR System 9700</td><td>Thermo Fisher Scientific</td><td>N805-0200</td><td></td></tr><tr><td>Dual Flat Block Sample Module</td><td>Thermo Fisher Scientific</td><td>4425757</td><td></td></tr><tr><td>QuantStudio 3D Tilt Base for Dual Flat Block GeneAmp PCR System 9700</td><td>Thermo Fisher Scientific</td><td>4486414</td><td></td></tr><tr><td>QuantStudio 3D Digital PCR Chip Adapter Kit for Flat Block Thermal Cycler</td><td>Thermo Fisher Scientific</td><td>4485513</td><td></td></tr><tr><td>QuantStudio 3D Digital PCR Chip Loader</td><td>Thermo Fisher Scientific</td><td>4482592</td><td></td></tr><tr><td>QuantStudio 3D Digital PCR Instrument with power cord</td><td>Thermo Fisher Scientific</td><td>4489084</td><td></td></tr></tbody></table>

detection of a cdh1 rare transcript variant in fresh-frozen gastric cancer tissues by chip-based digital pcr

CDH1a是CDH1基因的非规范化转录, 它被发现在一些胃癌 (GC) 细胞系中表达, 而在正常的胃粘膜中没有。最近, 我们在 GC 患者的新鲜冰冻肿瘤组织中检测到了CDH1a成绩单变体。本文采用基于芯片的数字 PCR (dPCR) 方法, 研究了该变异体在组织样品中的表达。dPCR 提供了一个准确的, 强健的, 高度敏感的核酸测量的潜力, 并越来越多地用于不同领域的应用。dPCR 能探测稀有目标;此外, dPCR 提供了绝对和精确的核酸定量的可能性, 不需要校准仪和标准曲线。事实上, 反应分区从背景中丰富了目标, 提高了放大效率和对抑制剂的耐受性。这样的特性使 dPCR 成为检测CDH1a稀有脚本的最佳工具。

使用这里介绍的过程, 我们检查了在胃新鲜冷冻组织中罕见的转录变体CDH1a的表达。dPCR 的分析是在21配对正常和癌症组织样本和11额外的肿瘤样本中进行的。CDH1a在 32 (47%) 个肿瘤中可检测到 15, 而没有正常的组织样本显示这一罕见的转录的存在5。在我们的分析中, 不到1.3万数据点的芯片被拒绝, 与非均质加载的芯片一样。图 1</s...

Watch this Scientific Journal Video about Detection of a CDH1 Rare Transcript Variant in Fresh-frozen Gastric Cancer Tissues by Chip-based Digital PCR at JoVE.com

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

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

该手稿描述了一种基于芯片的数字 PCR 方法, 用于检测从胃癌患者中获得的新鲜冰冻正常和肿瘤组织中罕见的CDH1转录变体 (CDH1a)。

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 ...

detection-cdh1-rare-transcript-variant-fresh-frozen-gastric-cancer

Universidad de Cartagena UDEC

Research

JoVE Journal

Cancer Research

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

6.2K 次观看.  Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS. 该手稿描述了一种基于芯片的数字 PCR 方法, 用于检测从胃癌患者中获得的新鲜冰冻正常和肿瘤组织中罕见的CDH1转录变体 (CDH1a)。

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

Development of a Human Preclinical Model of Osteoclastogenesis from Peripheral Blood Monocytes Co-cultured with Breast Cancer Cell Lines

The crosstalk between tumor cells and bone cells in the bone microenvironment is crucial to understanding the mechanism of bone metastasis formation. We developed an in vitro fully human preclinical model of a co-culture of breast cancer cells and monocytes undergoing differentiation towards osteoclasts. We optimized a model of osteoclastogenesis starting from a sample of peripheral blood collected from healthy donors. Peripheral blood mononuclear cells (PBMCs) were first separated by density gradient centrifugation, seeded at a high density and induced to differentiate by adding two growth factors (GFs): receptor activator of nuclear factor-&#954;B ligand (RANKL) and macrophage colony-stimulating factor (MCSF). The cells were left in culture for 14 days and then fixed and analyzed by downstream analysis. In osteolytic bone metastases, one of the effects of cancer cell arrival in bone is the induction of osteoclastogenesis. We thus challenged our model with co-cultures of breast cancer cells to study the differentiation power of cancer cells with respect to GFs. A straightforward way of studying cancer cell-osteoclast interaction is to perform indirect co-cultures based on the use of conditioned medium collected from breast cancer cell cultures and mixed with fresh medium. This mixture is then used to induce osteoclast differentiation. We also optimized a method of direct co-culture in which cancer cells and monocytes undergoing differentiation share the medium and exchange secreted factors. This is a significant improvement over the original indirect co-culture method as researchers can observe the reciprocal interactions of the two cell types and perform downstream analyses for both cancer cells and osteoclasts. This method enables us to study the effect of drugs on the metastatic bone microenvironment and to seed cell lines other than those derived from breast cancer. The model can also be used to study other diseases such as osteoporosis or other bone conditions.

This protocol describes development of an in vitro human preclinical model of osteoclastogenesis from peripheral blood monocytes cultured with breast cancer cell lines to mimic the cancer cell-osteoclast interaction. The model could be used to further our understanding of bone metastasis formation and improve therapeutic options.

乳腺癌细胞系外周血单核细胞破的人前临床模型的建立

The crosstalk between tumor cells and bone cells in the bone microenvironment is crucial to understanding the mechanism of bone metastasis formation. We ...

科研

癌症研究

Detection of Rare Mutations in CtDNA Using Next Generation Sequencing

The analysis of circulating tumor DNA (ctDNA) using next-generation sequencing (NGS) has become a valuable tool for the development of clinical oncology. However, the application of this method is challenging due to its low sensitivity in analyzing the trace amount of ctDNA in the blood. Furthermore, the method may generate false positive and negative results from this sequencing and subsequent analysis. To improve the feasibility and reliability of ctDNA detection in the clinic, here we present a technique which enriches rare mutations for sequencing, Enrich Rare Mutation Sequencing (ER-Seq). ER-Seq can distinguish a single mutation out of 1 x 107 wild-type nucleotides, which makes it a promising tool to detect extremely low frequency genetic alterations and thus will be very useful in studying disease heterogenicity. By virtue of the unique sequencing adapter&#39;s ligation, this method enables an efficient recovery of ctDNA molecules, while at the same time correcting for errors bidirectionally (sense and antisense). Our selection of 1021 kb probes enriches the measurement of target regions that cover over 95% of the tumor-related driver mutations in 12 tumors. This cost-effective and universal method enables a uniquely successful accumulation of genetic data. After efficiently filtering out background error, ER-seq can precisely detect rare mutations. Using a case study, we present a detailed protocol demonstrating probe design, library construction, and target DNA capture methodologies, while also including the data analysis workflow. The process to carry out this method typically takes 1-2 days.

This manuscript describes a technique for detecting mutations of low frequency in ctDNA, ER-Seq. This method is differentiated by its unique use of two-directional error correction, a special background filter, and efficient molecular acquirement.

利用下一代测序检测 CtDNA 中的稀有突变

The analysis of circulating tumor DNA (ctDNA) using next-generation sequencing (NGS) has become a valuable tool for the development of clinical oncology. ...

Serum and Plasma Copy Number Detection Using Real-time PCR

Serum and plasma cell free DNA (cfDNA) has been shown as an informative, non-invasive source of biomarkers for cancer diagnosis, prognosis, monitoring, and prediction of treatment resistance. Starting from the hypothesis that androgen receptor (AR) gene copy number (CN) gain is a frequent event in metastatic castration resistance prostate cancer (mCRPC), we propose to analyze this event in cfDNA as a potential predictive biomarker.
We evaluated AR CN in cfDNA using 2 different real-time PCR assays and 2 reference genes (RNaseP and AGO1). DNA amount of 60 ng was used for each assay combination. AR CN gain was confirmed using Digital PCR as a more accurate method. CN variation analysis has already been demonstrated to be informative for the prediction of treatment resistance in the setting of mCRPC, but it could be useful also for other purposes in different patient settings. CN analysis on cfDNA has several advantages: it is non-invasive, rapid and easy to perform, and it starts from a small volume of serum or plasma material.

This manuscript describes the copy number variation analysis performed in serum or plasma DNA using real-time PCR approach. This method is suitable for the prediction of drug resistance in castration resistant prostate cancer patients, but it could be informative also for other diseases.

应用实时 PCR 技术检测血清和血浆拷贝数

Serum and plasma cell free DNA (cfDNA) has been shown as an informative, non-invasive source of biomarkers for cancer diagnosis, prognosis, monitoring, ...

Establishment of a Primary Culture of Patient-derived Soft Tissue Sarcoma

Soft tissue sarcomas (STS) represent a spectrum of heterogeneous malignancies with a difficult diagnosis, classification, and management. To date, more than 50 histological subtypes of these rare solid tumors have been identified. Thus, due to their extraordinary diversity and low incidence, our understanding of the biology of these tumors is still limited. Patient-derived cultures represent the ideal platform to study STS pathophysiology and pharmacology. We thus developed a human preclinical model of STS starting from tumor specimens harvested from patients undergoing surgical resection. Patient-derived STS cell cultures were obtained from the surgical specimens by collagenase digestion and isolated by filtration. Cells were counted, seeded, and left for 14 days in standard monolayer cultures and then processed by downstream analysis. Before performing molecular or pharmaceutical analyses, the establishment of STS primary cultures was confirmed through the evaluation of cytomorphologic features and, when available, immunohistochemical markers. This method represents a useful tool 1) to study the natural history of these poorly explored malignancies and 2) to test the effects of different drugs in an effort to learn more about their mechanisms of action.

The following protocol focuses on the establishment of a primary culture of patient-derived soft tissue sarcoma (STS). This model could help us to better understand the molecular background and pharmacological profile of these rare malignancies and could represent a starting point for further research aimed at improving STS management.

病人衍生软组织肉瘤原代培养的建立

Soft tissue sarcomas (STS) represent a spectrum of heterogeneous malignancies with a difficult diagnosis, classification, and management. To date, more ...

Digital Polymerase Chain Reaction Assay for the Genetic Variation in a Sporadic Familial Adenomatous Polyposis Patient Using the Chip-in-a-tube Format

The quantitative analysis of human genetic variation is crucial for understanding the molecular characteristics of serious medical conditions, such as tumors. Because digital polymerase chain reactions (PCR) enable the precise quantification of DNA copy number variants, they are becoming an essential tool for detecting rare genetic variations, such as drug-resistant mutations. It is expected that molecular diagnoses using digital PCR (dPCR) will be available in clinical practice in the near future; thus, how to efficiently conduct dPCR with human genetic material is a hot topic. Here, we introduce a method to detect Adenomatous polyposis coli (APC) somatic mosaicism using dPCR with the chip-in-a-tube format, which allows eight dPCR reactions to be simultaneously conducted. Care should be taken when filling and sealing the reaction mixture on the chips. This article demonstrates how to avoid the over- and underestimation of positive partitions. Furthermore, we present a simple procedure for collecting the dPCR product from the partitions on the chips, which can then be used to confirm the specific amplification. We hope that this methods report will help promote the dPCR with the chip-in-a-tube method in genetic research.

Digital polymerase chain reaction (PCR) is a useful tool for the high-sensitivity detection of single nucleotide variants and DNA copy number variants. Here, we demonstrate key considerations for measuring rare variants in the human genome using digital PCR with the chip-in-a-tube format.

数字聚合酶链反应法测定零星家族性腺瘤性息肉病患者的基因变异

The quantitative analysis of human genetic variation is crucial for understanding the molecular characteristics of serious medical conditions, such as ...

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 ...

Genome-Wide Analysis of DNA Methylation in Gastrointestinal Cancer

DNA methylation is an important epigenetic change that is biologically meaningful and a frequent focus of cancer research. Genome-wide DNA methylation is a useful measure to provide an accurate analysis of the methylation status of gastrointestinal (GI) malignancies. Given the multiple potential translational uses of DNA methylation analysis, practicing clinicians and others new to DNA methylation studies need to be able to understand step by step how these genome-wide analyses are performed. The goal of this protocol is to provide a detailed description of how this method is used for the biomarker identification in GI malignancies. Importantly, we describe three critical steps that are needed to obtain accurate results during genome-wide analysis. Clearly and concisely written, these three methods are often lacking and not noticeable to those new to epigenetic studies. We used 48 samples of a GI malignancy (gastric cancer) to highlight practically how genome-wide DNA methylation analysis can be performed for GI malignancies.

Herein, we describe a procedure for genome-wide analysis of DNA methylation in gastrointestinal cancers. The procedure is of relevance to studies that investigate relationships between methylation patterns of genes and factors contributing to carcinogenesis in gastrointestinal cancers.

胃肠癌DNA甲基化基因组全谱分析

DNA methylation is an important epigenetic change that is biologically meaningful and a frequent focus of cancer research. Genome-wide DNA methylation is ...

使用 Ion Torrent 测序的 SNP 指导的胃癌 5-FU 疗法

Multi-Gene Single Nucleotide Polymorphism Detection in Gastric Cancer Based on Ion Semiconductor Sequencing Platform

Gastric cancer is a common heterogeneous tumor. Most patients have advanced gastric cancer at the time of diagnosis and often need chemotherapy. Although 5-fluorouracil (5-FU) is widely used for treatment, its therapeutic sensitivity and drug tolerance still need to be determined, which emphasizes the importance of individualized administration. Pharmacogenetics can guide the clinical implementation of individualized treatment. Single nucleotide polymorphisms (SNPs), as a genetic marker, contribute to the selection of appropriate chemotherapy regimens and dosages. Some SNPs are associated with folate metabolism, the therapeutic target of 5-FU. Methylenetetrahydrofolate reductase (MTHFR) rs1801131 and rs1801133, dihydrofolate reductase (DHFR) rs1650697 and rs442767, methionine synthase (MTR) rs1805087, gamma-glutamyl hydrolase (GGH) rs11545078 and solute carrier family 19 member 1 (SLC19A1) rs1051298 have been investigated in different kinds of cancers and antifolate antitumor drugs, which have potential forecasting and guiding significance for application of 5-FU. The ion torrent next-generation semiconductor sequencing technology can rapidly detect gastric cancer-related SNPs. Each time a base is extended in a DNA chain, an H+ will be released, causing local pH changes. The ionic sensor detects pH changes and converts chemical signals into digital signals, achieving sequencing by synthesis. This technique has low sample requirement, simple operation, low cost, and fast sequencing speed, which is beneficial for guiding individualized chemotherapy by SNPs.

作者聚焦：胃癌氟尿嘧啶反应的遗传分析

This protocol proposes holistic laboratory procedures required to detect single nucleotide polymorphisms in gastric cancer samples based on an ion semiconductor sequencing platform. The target sequences, ligating adapters, library amplification and purification, and quality control criteria are also described in detail.

基于离子半导体测序平台的胃癌多基因单核苷酸多态性检测

Gastric cancer is a common heterogeneous tumor. Most patients have advanced gastric cancer at the time of diagnosis and often need chemotherapy. Although ...

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

摘要

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此视频中的章节

乳腺癌细胞系外周血单核细胞破的人前临床模型的建立

利用下一代测序检测 CtDNA 中的稀有突变

应用实时 PCR 技术检测血清和血浆拷贝数

病人衍生软组织肉瘤原代培养的建立

数字聚合酶链反应法测定零星家族性腺瘤性息肉病患者的基因变异

患者生物流体中肿瘤相关循环DNA的检测与监测

胃肠癌DNA甲基化基因组全谱分析

基于离子半导体测序平台的胃癌多基因单核苷酸多态性检测

基于离子半导体测序平台的胃癌多基因单核苷酸多态性检测

基于离子半导体测序平台的胃癌多基因单核苷酸多态性检测