我们感谢我们的合作者, 斯蒂芬. 琼斯, 查林顿, 戴安娜博士和来自数字生物学中心 (生物 Rad 公司 CA) 的萨曼莎. 库珀博士, 他们的化验设计支持;Nezar Rghei 和 Moemen 博士 Abdalla (Norgen Biotek, 加拿大) 在优化 RNA 提取协议的关键建议;和香农坎贝尔, 斯科特瑟斯顿, 杰夫 Fensterer, 香农马尔泰洛塔和 Joellyn Enos 协助的测试要求和商业监测。

除非另有说明, 否则制造商的指示将遵循下面列出的试剂。PCR 检测是商业上可用的产品, 旨在探测 ROS1 和 RET 融合。
1. 与 RNA 一起在准备反向转录 (RT)-dPCR: 实验室最佳做法
<ol><li>在使用 RNA 时创建一个无 RNase 的环境。<ol>
<li>使用可用于灭活污染 RNases 的商用喷雾剂。</li>
		<li>使用经认证的无 RNase 试剂、小贴士和试管。使用障碍提示 pipettors, 以防止引入 RNases 或交叉污染的样品。</li>
	</ol>
</li>
	<li>总是穿上实验室的外衣, 以防微粒从衣物中掉落到样品中。指定用于 RNA 处理的实验室大衣。</li>
	<li>戴上手套, 防止皮肤 RNases 的样品污染。经常更换手套。 注意:假设实验室表面受到 RNase 的污染, 因为它们暴露在环境中。手套, 接触皮肤, 头发, 面糊, 冷冻手柄, 钢笔/标记,等被假定为不再 RNase。</li>
	<li>使用前用 RNase 灭活喷雾净化 pipettors、benchtops、离心机和其他工作表面。</li>
	<li>如果可能的话, 维护一组与 RNA 一起使用的设备。</li>
	<li>在使用 RNA 样品时尽量减少实验室区域内气流的中断, 以防止微粒落入样品或污染工作区域。</li>
	<li>将纯化的 RNA 贮存在-80 ˚C。</li>
	<li>避免多次冰冻融化的 RNA 样本, 因为这可能会导致退化。</li>
</ol>2. 用于阳性对照的分析 RNA 材料的生成
<ol><li>使用已发布的 mRNA 序列为感兴趣的融合变体设计合成 DNA10。<ol>
<li>对于给定的融合变体, 选择一个 mRNA 融合序列, 其中包括融合站点加上足够长的侧翼两侧, 以覆盖 PCR 扩增子。</li>
		<li>选择 50-250 nt 之间的核苷酸序列, 以模拟利用血小板富集等离子体捕获的循环 RNA 的大小。</li>
		<li>将 T7 启动程序序列 (5 '-CAGAGATGCATAATACGACTCACTATAGGGAGA-3 ') 添加到目标序列的 5 ' 端。</li>
	</ol>
</li>
	<li>将合成序列作为双绞链脱氧核糖核酸 (DNA) 片段排序。</li>
	<li>将三 EDTA (TE) 缓冲器中的合成 DNA 重组为最终浓度为10µL。</li>
	<li>使用体外转录将 60 ng 合成 DNA 转换为 RNA。</li>
	<li>用苯酚/胍基试剂纯化 RNA 记录12。<ol>
<li>包括 DNase I, 无 RNase 去除残留模板 DNA。</li>
	</ol>
</li>
	<li>使用具有 rna 特异染料和标准的商用荧光计测量纯化的体外 RNA 的浓度。确保 RNA 位于所选标准所能接受的范围内。可能需要稀释。</li>
	<li>用2% 琼脂糖凝胶混合 rna 凝胶染色和高射程 rna 阶梯, 包括 50-250 nt 大小范围, 确认成功的转录。<ol>
<li>将每个体外RNA 的 500 ng 加载到凝胶上。</li>
		<li>用5伏/厘米的凝胶。</li>
		<li>使用光照可视化单个波段, 并记录结果。</li>
		<li>确认每个融合变体的预期成绩单大小 (基于步骤2.1.2 中的设计)。</li>
	</ol>
</li>
	<li>使用匹配的变异特异 PCR 方法确认 dPCR 检测每个的体外RNA (参见本协议的步骤 5-8)。</li>
	<li>可选: 准备包含每个融合变体和控制基因 GUSB 的体外RNA 的摩尔混合物。</li>
	<li>如果执行了步骤 2.9: 通过 dPCR 使用变体特定的 PCR 方法确认检测控制混合物中包含的每个融合变体 (参见本协议的步骤 5-8)。</li>
	<li>通过测试0.25 到 2.5 fg10的浓度, 确定分析阳性控制所需的输入浓度。根据所需的复制号输出选择浓度。</li>
	<li>确认后, 准备10µL 单使用整除数的分析 RNA 用于阳性控制 (步骤 4.4) 和存储在-80 ˚C。</li>
</ol>3. 捐助者标本
<ol><li>收集10毫升的人全血标本在10毫升血液收集管 (BCT) 含有无细胞 RNA 防腐剂。 注意:所有人类捐助者均应同意研究使用, 在测试期间不得收集或使用捐助者特定的识别信息。</li>
	<li>在 BCT 制造商指定的时间范围内处理整个血液样本。</li>
	<li>汇集正常人血浆可以从商业来源购买在分析的正面控制之内使用。准备单一用途, 1 毫升整除数的汇集正常人血浆并且存放在-80 ˚C 为使用与正面控制 (步 4.4)。</li>
</ol>4. 从等离子体中回收循环 RNA
注意: 在这个过程中快速工作是很重要的。
<ol><li>离心机全血管以 200 x g 为20分钟。</li>
	<li>使用血清学吸管从离心采血管收集多达4毫升的血浆。小心不要打扰或吸入巴菲大衣层。</li>
	<li>使用可从血浆中捕获外来体、血小板和无细胞 rna 的商用试剂盒分离循环 rna。将 RNA 从阳性对照样品中分离出来。</li>
	<li>
为每批临床样品准备积极的控制, 如下所示:
	<ol>
<li>解冻1毫升的正常人血浆整除 (步骤 3.3)。</li>
		<li>解冻10µL 分析 RNA 整除 (步骤 2.12)。</li>
		<li>当乙醇加入到血浆裂解液中后, 将10µL 分析 RNA 添加到正常的人体血浆样品中, 准备阳性控制。</li>
	</ol>
</li>
	<li>洗脱样品与100µL 核酸酶无水。立即进行 RNA 清理和浓缩。<ol>
<li>样品可以储存在湿冰上, 并覆盖, 长达一小时。</li>
	</ol>
</li>
	<li>用柱基法和洗脱在9µL RNase 水中浓缩 RNA。<ol>
<li>立即进行步骤 5, 或将样品放在湿冰上长达一小时。</li>
	</ol>
</li>
</ol>5. RNA 的反向转录到 cDNA
<ol><li>使用商业上可用的反向转录反应套件 (包括随机引物) 将浓缩循环 RNA 样本转化为 cDNA (请参见表 1中的组件)。 注意:基因特异引物是可选的, 可以设计为测试变种。引物是根据目标 RNA 序列设计的。使用步骤2.1 中的融合变体序列。<ol>
<li>不包括逆转录酶控制样本和没有 RNA 控制样本 (请参见表 1)。</li>
	</ol>
</li>
	<li>利用商业上可用的 DNA 浓缩器自旋柱将 cDNA 从反向转录反应中分离出来。 注意:这一步骤有助于去除酶, 底漆和自由核苷酸 triphosphates (dNTPs)。</li>
	<li>立即使用 cDNA PCR 反应或存储在-80 ˚C。</li>
</ol>6. 数字 PCR
注: 此 pcr 是特定的水滴数字 pcr (见材料表)。
<ol><li>
PCR 混合预防措施。
	<ol>
<li>穿上一次性的实验室大衣和丁腈橡胶手套。</li>
		<li>在专用试剂制备区使用 PCR 混合试剂。请勿在试剂制剂区处理 cDNA。</li>
		<li>在工作时覆盖探头以保护它们不受光线的侵害。过量的光可以照片漂白的荧光染料附着在探针上。</li>
		<li>运输混合, 覆盖和保护, 从光, 到一个单独的预放大区域之前, 将添加 cDNA。</li>
		<li>添加 cDNA, 以 pcr 混合在 pcr 清洁罩位于预放大区域。</li>
	</ol>
</li>
	<li>根据表 2, 为20µL 的最终反应量准备 PCR 混合。</li>
	<li>将 pcr 混合 + cDNA 分发到 pcr 板上。 注意:建议使用板布局作为参考。</li>
	<li>使用可拆卸板封口机盖板。</li>
	<li>离心板简单地收集在井底的样品。</li>
	<li>混合在平板振动筛在低设置十年代。</li>
	<li>离心板简单地收集井底的样品。</li>
	<li>拆卸板封口机。使用手动或自动雾滴生成系统进行 PCR cDNA 混合的雾滴生成。<ol>
<li>对于手动滴产生, 转移20µL PCR 混合到样品井上的水滴产生墨盒。加入70µL 的液滴生成油。用橡皮垫片和转移墨盒盖上手动滴发生器, 以启动雾滴生成。跟随滴世代, 转移滴到一个新的 PCR 板材使用制造商推荐的提示。吸入和分配的水滴缓慢, 超过5至 6, 每一个, 而不接触的尖端开口的水滴墨盒或板块。</li>
		<li>对于自动液滴生成, 用箔密封密封板并将其转移到液滴发生器。确保所有的提示, 墨盒和板块在开始滴产生之前到位。</li>
	</ol>
</li>
	<li>跟随水滴的产生和转移到一个新鲜的 PCR 板, 密封与箔板封口机, 和热循环板使用设置在表 3。</li>
	<li>在热循环仪运行完成后, 用滴读器读取该板。创建用于识别控件、示例、等位置的读取器软件的版面布局, 并将加载到软件中以开始读取。</li>
</ol>7. 数据分析和审查以及成果的产生
<ol><li>使用商用软件分析板读结果。</li>
	<li>导航到 "分析" 菜单以查看二维 (2D) 振幅图。</li>
	<li>通过检查水滴数据来评估数据的整体质量。<ol>
<li>使用 "事件" 菜单计算总接受事件编号的数据。如果每个井都少于1万个事件, 请仔细评估数据以了解其他问题。</li>
		<li>检查数据的异常荧光振幅。复制样本之间的显著振幅差异和浓度差异表明, 样品处理或混合不畅。</li>
		<li>在45度轴上用喷雾模式制作水滴簇的注释, 这表明质量差的水滴或有问题的样品。</li>
		<li>检查阳性对照, 无逆转录酶 (无 RT), 无 RNA 控制 (NRC) 数据优先。选择所有的控制样本, 并通过2D 图检查集群质量。对于适当的阈值, 液滴簇之间的清晰分离应该是明显的。</li>
	</ol>
</li>
	<li>对于每个检测变量, 设置基于控制井的阈值。<ol>
<li>使用十字线工具在2D 地块上设置阈值, 将双负滴种群从控制基因种群中分离出来 (标记为 5 '-hexachloro 荧光素铈 phosphoramidite 探针)、y 轴和变异基因种群 (如果存在) (标有荧光素 amidite 或 6-羧基探针), x 轴。</li>
		<li>向单个示例中的每个复制的副本求和。</li>
		<li>以检测到的变体副本数量表示测试结果。 注意:要确定用于调用阳性或阴性样本的分析截止值, 请运行一个正常的健康捐献者样本集 (至少10个样本), 并通过最后确定的过程建立截止到任何可检测的背景信号的突变兴趣。另外, 建立控制基因拷贝所需的数量来调用一个积极或消极的结果。这种控制基因切断功能作为内部质量控制 (QC), 以评估每一个 RNA 样品的数量和质量的处理。</li>
	</ol>
</li>
</ol>8. 使用细胞线验证 RT-dPCR 反应条件 (可选)
<ol><li>为了验证融合变种的检测, 使用商业上可用的细胞线表达 ROS1 或 RET 融合 mRNA 的兴趣。按如下方式进行:<ol>
<li>融汇在胍的溶解溶液中, 直接从冷冻状态中的闪光冷冻细胞。即使在均匀化之前的短暂解冻也会导致 RNA 的降解和损耗。</li>
		<li>使用为 rna 设计的硅膜自旋柱分离 rna。</li>
		<li>使用带有 rna 特异试剂和标准的荧光计测量 rna 样品的浓度。</li>
		<li>将分离的 rna 稀释成来自等离子体或其他商业来源的野生型 rna 的背景。</li>
	</ol>
</li>
	<li>从 RNA 的反向转录到 cDNA、数字 PCR、数据分析和回顾以及在本协议中列出的结果的生成, 以确认所需的变种的检测步骤。</li>
</ol>

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罗杰、K.A. 和 G.A.P. 是 Biodesix、罗杰和 G.A.P. 的雇员, 并持有 Biodesix 的专利申请的共同发明者, 涵盖了检测非小细胞循环遗传变异的诊断测试系统。肺癌。

RET 和 ROS1 重排在一起弥补了3% 的驱动突变在 NSCLC 人口18。虽然罕见, 但这些基因改变的检测是至关重要的。非肺癌患者的这些改变可能得益于针对性的治疗, 特别是抑制异常激酶活动的结果, 从 onco 蛋白13。一些这样的疗法已经被 FDA 批准用于 ROS1 阳性的 NSCLC, 而其他一些已经被证明是有效的抗 RET 在临床试验19。
数字 PCR 技术为液体活检应用提供了最理想的灵敏度20。这项技术已被广泛应用于无细胞 DNA 的循环使用, 用于测量非 NSCLC 患者的肿瘤突变4,6,21,22,23.除了 cfDNA, 我们还开发了一项协议, 旨在强健地测量来自循环肿瘤 RNA (图 1a)10的 NSCLC 患者中最流行的融合变体。
我们建立的协议允许检测的分析限制降低到 0.2% (图 2)。虽然 dPCR 是特别具体和敏感的, 测试是有限的已知融合变种的面板被选择和多路检测在 PCR 试验。因此, 必须仔细选择融合在复用化验中, 以确保适当的覆盖范围内的肺癌患者的人群。我们成功地设计了用于 ROS1 的测试, 同时检测到八个融合变体是由 ROS1 位点的重排和99% 和88% 的 ret 和 ROS1 阳性人口分别 (图 1B-C)17。
本研究中描述的最终测试工作流包括批处理控件, 以确保结果的一致性。这些控制包括一个正分析标准和两个负控制, 它们一起确保在批处理中不会发生污染或 PCR 抑制 (图 3)。为了确保检测的健壮性, 在21天期间使用批处理控制进行了一项研究 (图 3D、H)。这些数据显示了在本协议中建立的 RNA 过程的一致性。
良好的实验室实践和适当的 RNA 处理是确保可靠和准确结果的关键组成部分。实验室空间和设备专用于使用 RNA, 每次使用后清洗设备, 使用无 RNase 试剂和消耗品, 并将 RNase 灭活喷雾应用于工作空间, 都有助于减少污染 RNases。由技术员认真处理 rna 样品, 包括专门的实验室大衣, 频繁的手套变化, 迅速通过 RNA 提取程序, 并保持样品在冰上是至关重要的保存样本完整性。一旦 RNA 被反向转录为 cDNA, 样品是在一个更稳定的形式, 不容易退化。除了支持 RNA 完整性的做法外, 还应在隔离区域保持 PCR 成分和样本, 以防止交叉污染, 从而导致假阳性结果。聚合 pcr 试剂和 pcr 母料的制备应与 pcr 模板分离, 并应非常小心地将扩增模板 (后 pcr) 与所有预扩材料 (包括试剂、RNA 和 cDNA 样本) 隔离开来。最后, 在放大前对乳化 PCR 混合物进行适当的生成和处理是保持液滴完整性和最佳 dPCR 条件的关键。这样的预防措施在执行本协议时非常关键, 以获得一致和准确的结果。所有数据应由训练有素的人员在公布结果之前进行检查, 以确保所有 QC 指标都得到满足。如果是次优结果 (图 4), 则必须由技术人员和实验室主任审查批次, 并可能需要重新处理。
dPCR 结果可以提前24小时产生, 从样品收据和95% 的样本结果在本研究所使用的测试集 (n = 984) 是报告到订购医生在不到72小时从收据 (图 5)。这个转过来的时间为医生提供了需要的分子信息在一个时间框架, 允许启动适当的治疗。这些结果通常比使用常规组织活检获得的更早。可利用类似的基于 RNA 的方法发展非肺癌和其他癌症的其他生物标志物, 并将从同样迅速的结果中获益。例如, 使用 RT dPCR 对程序性死亡配体 1 (PD-L1) mRNA 转录进行测量可以向医生通报免疫治疗的选择。对液体活检和 dPCR 在监测治疗效果方面也有越来越浓厚的兴趣。早期的迹象表明, 肿瘤的复发, 使用基因组检测的特定变种, 可以让医生调整治疗方案之前, 病人的症状的护理标准, 如成像24。这项研究中报告的协议, 由于它们的非侵入性、灵敏度、快速转接时间和成本效益而成为监测的理想选择。本文所述的化验结果在72小时内提供了从样本收据, 以最低的假阳性检测率, 这有助于快速治疗决策, 并绕过一些限制, 以组织为基础的测试4。
我们的协议和数据显示了一个可靠的测试系统, 用于鉴别低丰度 RNA 变种, 以及在临床实践中血液变异检测的潜力。对于那些没有通过快速靶向液体活检方法识别的驾驶员突变的患者, 从组织和血液中添加更广泛的基因组和蛋白质蛋白可以提供更广泛的临床信息。支持治疗计划。

25% 的非小细胞肺癌 (NSCLC) 患者在诊断时可能没有足够的组织可供测试。即使在组织可用的情况下, 也可能没有足够的数量或质量来执行推荐的分子测试1,2。如果有足够的组织从活检的分子分析, 病人可能需要等待数周或更长的结果, 或开始治疗没有分子结果3,4。然而, 考虑到多目标治疗方案的出现, 对于被诊断为 NSCLC 的患者, 重要的是提供信息性的分子诊断。从液体活检中检测循环无细胞 DNA (cfDNA) 是解决传统组织测试的挑战的方法4,5,6。目前的可操作性突变的测试选项使用 cfDNA 和类似的 dPCR 工作流快速生成, 包括表皮生长因子受体 (egfr) 敏化突变ΔE746 A750 和 L858R, EGFR 抵抗突变 T790M, KRAS 原癌基因 (KRAS) 变种和 B 皇家空军原癌基因 (BRAF) 变种 V600E。虽然没有广泛采用的领域, 循环肿瘤衍生信使 RNA (mRNA) 分离的液体活检也可以提供重要的临床信息7,8,9。我们以前已经开发并报告了多路检测棘皮动物微管相关蛋白的方法, 如 4-变性淋巴瘤受体酪氨酸激酶 (EML4-ALK) 融合变种的血浆10。在本研究中, 我们将这些方法扩展为 ROS1 和 RET 的高阶多路复用 RNA 靶, 在每种检测中覆盖八个融合变种。目的是开发一种快速、灵敏、特异和可重复的技术, 用于检测先前诊断为 NSCLC 的患者血浆中的这些融合变种。
测试过程是在医生的办公室开始使用 RNA 稳定血液收集管11。这些管子含有细胞防腐剂以及 RNase 抑制剂。样品在一夜之间被运输优先权对美国病理学家集中的学院 (盖帽)-认可的或临床实验室改善修正 (CLIA)-被证明的实验室 (临床实验室) 为处理由能干的人员。一旦得到临床实验室, 每一个步骤的处理是根据批准的标准操作程序 (SOP) 进行。全血是离心恢复血浆, 然后用于隔离循环 RNA 是免费的血液或在封装基团, 如外来体和血小板 7, 8, 9.为了将 rna 从这些隔间分离出来, 我们选择了一种基于几种提取方法比较的 rna 恢复系统。分离 RNA 是集中和反向转录到 cDNA。在优化 cDNA 合成方法的过程中, 对几种逆转录酶和基因特异引物进行了评价, 以最大限度地提高 ROS1 和 RET 靶转录的转化率10。这对于低丰度的循环转录, 如肿瘤衍生的融合变种是至关重要的。最后, 我们优化 dPCR 底漆和探针浓度, 以允许多路检测的 RET 或 ROS1 融合变种和控制基因, glucuronidase β (GUSB)。然后, 在执行本报告所述的分析验证研究之前, 我们将每个优化研究的最佳条件组合为最终锁定的协议。该协议和这些结果为快速而敏感的工作流程提供了基础, 用于常规检测循环中的稀有融合变体。

a blood-based test for the detection of ros1 and ret fusion transcripts from circulating ribonucleic acid using digital polymerase chain reaction

我们开发了一种新的方法来分离和鉴定肿瘤衍生循环核糖核酸 (茨尔纳) 的血液为基础的液体活检。从血液中恢复的茨尔纳的稳健检测代表了一种解决临床诊断中关键未满足需求的方法。该测试始于收集血液收集管含有防腐剂, 稳定茨尔纳。在这个测试系统中, 从血浆中分离出无细胞、exosomal 和血小板相关的 RNA。茨尔纳是反向转录为补充 DNA (cDNA) 和扩增使用数字聚合酶链反应 (dPCR)。对目标生物标志物以及控制基因的样品进行评估。测试验证包括检测的限制、准确性和分析样本的鲁棒性研究。这项研究开发的方法重现性检测多融合变种为 ROS1 (C-Ros 原癌基因 1; 8 变种) 和 RET (重新排列在转染原癌基因; 8 变种)。对示例处理工作流进行了优化, 使测试结果可以在样本收据72小时内持续生成。

本协议描述了一个测试系统, 用于检测 RNA 融合变体, 用于测量 NSCLC 患者血浆中的驱动突变 (图 1a)。融合 mRNA 产品从最常见的 RET 和 ROS1 重排的表达在 NSCLC 人口被确定了13,14,15,16,17。然后设计了多路 PCR 检测方法, 以在单个反应中发现 NSCLC 中每个靶的八种最常见的转录变异。ROS1轨迹中最常见的易位生成与CD74、 SDC4、 SLC34A2、 EZR或TPM3基因 (图 1B) 的 5 ' 部分的关联。RET轨迹中最常见的易位导致与KIF5B并列, 该检测涵盖六个外显子结点。包括CCDC6和TRIM33 (图 1C) 的其他RET合作伙伴。总共, 该化验涵盖约88% 的 ROS1 和99% 的 RET 改变已知发生在 NSCLC 患者人口17。
首先使用八个单独的体外rna 对检测成分的特异性进行评估, 其中包含了 ROS1 或 RET 复用检测所覆盖的融合记录的 mRNA 序列。每个 RNA 物种都对每种不同的变异检测进行了测试, 包括复用版本。这些化验没有交叉反应性, 因此在设计的多路复用化验 (未显示的数据) 中展示了100% 分析特异性。为了确定测试协议检测的下限, 从表达融合变体的细胞系中提取的总 rna 在5%、1%、0.2% 和0.04% 浓度下混合成正常 RNA 的背景。多路复用和 ROS1 变异 PCR 方法检测到多达0.2% 融合变体 (图 2A B)。此外, 未通过多路 ROS1 和 RET 分析检测出5% 个离靶细胞线衍生 RNA (表达 EML4-ALK 融合转录) 的制备, 进一步证明特异性 (图 2a B)。
对 ROS1 和 dPCR 工艺进行了精密测试. 由摩尔体外rna 组成的分析控制材料, 通过反向转录和 dPCR 对三进行三浓度 (高、中、低) 的处理。在同一天内的不同场合 (天内), 连续三天 (日间), 并与两名经营者 (互操作者)。精密测试的结果表明, 准确检测的融合记录的兴趣, 以及控制基因, GUSB, 其中包括作为内部 QC 指标 (图 2C-D)。
除了 GUSB 内部控制外, 每批临床样品都使用一组批次控制来运行。一个积极的控制是从一个分析性的体外rna 的混合物, 代表每一个融合变种测试在 RT-dPCR, 以及分析体外RNA 为 GUSB。在 rna 提取过程中, rna 被注入正常的人体血浆裂解物中, 并在整个协议中与临床样品一起处理。无逆转录酶 (无 RT) 控制是一种阴性控制, 以确认在 rna 提取工作流中没有污染物质, 并证明了 rna 引物的特异性。无 RT 控制是使用与阳性对照相同的材料生成的, 但不包括在 cDNA 合成反应中的酶。无 RNA 控制 (NRC) 是一个阴性的控制, 以确认没有污染记录在反向转录反应成分。该控制被引入到工作流中的 cDNA 合成步骤, 和水添加到反应, 而不是 RNA 模板。如果要传递准确的结果, 则两个通道中的无 RT 和 NRC 控件必须为负值。表 1列出了每个控件的反向转录反应组件。为 ROS1 (图 3 -c) 和 RET (图 3 E G) 复用分析显示了每个控件的2D 图形的示例。使用荧光素 amidite (FAM) 探针检测融合变体, 并以 y 轴表示, 而控制基因 GUSB 则使用 5 '-hexachloro 荧光素 CE phosphoramidite (十六进制) 探针检测, 并位于 x 轴上。这些批次控制在21天的过程中进行评估, 以确定检测的鲁棒性。在研究过程中执行的所有21运行中, 均观察到融合阳性液滴和 GUSB 控制基因滴, 用于 ROS1 和 RET (图 3D, H)。所有负控制 (无 RT 和 NRC) 在整个21天内产生负结果 (未显示数据)。
解决问题的能力是在临床实验室设置中运行的任何测试协议的关键组成部分。在这里, 我们提供了使用 RT dPCR 协议的子优化结果的真实世界示例。第一个例子是2D 图, 展示了无逆转录酶控制的重要性 (图表 4A)。在这个例子中, 即使由于缺乏酶而没有 cDNA 转换, 突变阳性滴也存在。这一结果很可能是由于 dPCR 底漆放大了目标基因组 DNA。在这种情况下, 设计一个内含子生成的检测将防止基因组 DNA 的扩增。另外, RNase 的 DNase 酶可以用来消除污染的 DNA, 但这并不推荐用于检测稀有靶点, 因为某些 RNA 降解可能发生在与酶的孵化过程中。下一个示例2D 绘图是在两个通道中具有正水滴的 NRC (图 4B)。这表明在 dPCR 设置中的某个点有污染。在这种情况下, 建议放弃任何可能被污染的试剂用于测试, 彻底清除所有设备, 并重新测试新的反应成分。第三个示例2D 图, 显示为沿45°线的水滴喷雾 (图 4C)。这通常是由水滴的剪切和凝聚引起的。在热循环之前小心滴处理是必不可少的, 因为水滴容易损坏。我们建议在可用时使用自动雾滴生成。如果手动转移生成的水滴, 一定要选择建议的宽孔提示, 并采用仔细的吹打技术。滴转移需要缓慢的吸入和配药, 每次发生超过5-6 秒, 这是必不可少的吸管尖端打开不接触滴墨盒或良好。在配药时, 将吸管尖端保持在液位上, 并随着水滴的排出而缓慢升高 (查看视频以供演示)。最后的2D 绘图示例演示了正和负雾滴群之间缺少分离 (图 4D)。这可能有几个原因。强 PCR 抑制剂, 如用于裂解缓冲液的洗涤剂和过量的高降解 DNA, 可能导致分离的丧失。在这种情况下, 考虑在 cDNA 合成和 dPCR 之间添加一个清理步骤 (如本协议步骤5中所述)。最后, 缺乏分离也可能是由于次最优放大条件, 同时也应考虑 PCR 步骤的优化。
图 5中的数据代表了984个真实的世界患者采样次数, 并演示了此测试工作流的快速特性。结果在72小时内, 早在48小时 (79% 例) 的样品收据和95% 的病例报告给治疗医生。总之, 使用稳定的循环 rna 采血管, 优化的 rna 提取程序从血液和 RT dPCR 运行根据一个优化的协议与适当的内部和批次控制, 可以提供一个快速测试系统与 NSCLC 相关的融合 RNA 变异的准确检测。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57079/57079fig1v2.jpg"> 图 1: 使用特定于 NSCLC 中最常见的 RET 和 ROS1 变体的检测方法, 概述了用于融合变体探测的血液样本处理步骤.(A) 在绘制全血并将标本收集工具包中的 BCT 运送到临床实验室时, 启动样本测试。循环 RNA 从血小板丰富的血浆中的多个来源恢复, 反转转录与基因特异性启动, 并纯化, 用于 dPCR。样品使用一个商业上可用的系统来处理, 它由水滴生成 (乳液)、放大和水滴计数组成。数据使用商用软件进行分析。测试结果将被记录下来并报告给测试要求的医生。该过程设计为在72小时的时间内工作, 从样本收据到结果发布。八变种为 (B) ROS1 和 (C) RET 在复用化验之内包括。由 Biodesix 网站改编而成。<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57079/57079fig1v2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57079/57079fig2.jpg"> 图 2: 分析验证.细胞线表达 (A) SDC4-ROS1 融合和 (B) CCDC6-RET 融合被稀释的背景下, 总人类野生型 rna (野生 rna)。每一个融合变体, 检测的限制, 建立在0.2% 的变化频率使用预先确定的标准, 每种变异检测。上述阈值之上的所有样本还包含至少21个控制基因拷贝。对野生型 RNA 背景下的 5% EML4-ALK (音译) 标准进行了测试, 以证明检测特异性, 结果证实为阴性。在高、中、低浓度下测量了分析复用 RNA 标准 (C) ROS1 和 (D) RET. 在同一天 (天内) 进行了三次以上的测试, 三次连续三天 (白天) 运行, 并与两个独立运算符 (互操作符)。显示了复制号和标准偏差的方法。由 Biodesix 网站改编而成。<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57079/57079fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57079/57079fig3.jpg"> 图 3: 批处理控制示例和健壮性数据.2D ROS1 复用分析 dPCR 结果为 (A) 阳性控制, (B) 无逆转录酶控制, (C) 没有 RNA 模板控制。(D) 控件连续21天运行 (不包括周末和节假日)。平均复制号 +/-标准偏差为 ROS1 正控制是 439 +/-141。没有逆转录酶, 也没有模板控件每天都运行, 这些都是阴性 (没有显示数据)。2D. dPCR 复用分析图的计算结果为 (E) 正则控制, (F) 无逆转录酶控制和 (G) 没有 RNA 模板控制。(H) 控件连续21天运行 (不包括周末和节假日)。平均拷贝 +/-标准偏差为 RET 正控制是 586 +/-182。没有显示的是没有逆转录酶和没有模板控件, 也在每天运行, 都是阴性的。由 Biodesix 网站改编而成。<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57079/57079fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57079/57079fig4.jpg"> 图 4: 诊断 RT dPCR.2D 图, 表示在无逆转录酶控制范围内 (A) 污染的情况下获得的次级最佳 dPCR 结果, (B) 在无 RNA 控制范围内的污染 (C) 剪切和凝聚的水滴, 和 (D) 优化的 pcr 条件较差或 pcr 抑制。<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57079/57079fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/57079/57079fig5.jpg"> 图 5: 周转时间 (达).对请求 RNA 变体 (n = 984) 的测试编译了达 (小时)。数据不包括周末、节假日和为 > 24 小时举行的样本, 原因是实验室测试请求表上的临床信息不完整。<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57079/57079fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Table 1" src="/files/ftp_upload/57079/57079table1.jpg"> 表 1: 制备过程控制用反向转录试剂。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>组件</td>
			<td>体积</td>
		</tr>
<tr>
<td>2x dPCR supermix 探头 (no 2 '-脱氧尿苷 5 '-三磷酸)</td>
			<td>10µL</td>
		</tr>
<tr>
<td>20x 可变目标引物/探头集 (450 nmol/升底漆, 250 nmol/升探头)</td>
			<td>1µL</td>
		</tr>
<tr>
<td>20x 控制目标引物/探头集 (450 nmol 底漆, 250 nmol/升六角探针)</td>
			<td>1µL</td>
		</tr>
<tr>
<td>核酸酶水</td>
			<td>1µL</td>
		</tr>
<tr>
<td>Cdna</td>
			<td>7µL</td>
		</tr>
</tbody></table>表 2: 准备 dPCR 的主混合料。
<table border="1" fo:display-align="center" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>循环步骤</td>
			<td>温度</td>
			<td>时间</td>
			<td># 周期</td>
			<td>坡道率</td>
		</tr>
<tr>
<td>酶活化</td>
			<td>95°c</td>
			<td>10分钟</td>
			<td>1</td>
			<td rowspan="4">~ 2 oc/s</td>
		</tr>
<tr>
<td>变性</td>
			<td>94°c</td>
			<td>三十年代</td>
			<td rowspan="2">40</td>
		</tr>
<tr>
<td>退火/延伸</td>
			<td>55°c</td>
			<td>1分钟</td>
		</tr>
<tr>
<td>酶失活</td>
			<td>98°c</td>
			<td>10分钟</td>
			<td>1</td>
		</tr>
<tr>
<td>保持 (可选)</td>
			<td>4°c</td>
			<td>无限</td>
			<td>1</td>
			<td>~ 1 oc/s</td>
		</tr>
</tbody></table>表 3: 热循环条件。

Watch this Scientific Journal Video about A Blood-based Test for the Detection of ROS1 and RET Fusion Transcripts from Circulating Ribonucleic Acid Using Digital Polymerase Chain Reaction at JoVE.com

A Blood-based Test for the Detection of ROS1 and RET Fusion Transcripts from Circulating Ribonucleic Acid Using Digital Polymerase Chain Reaction

用数字聚合酶链反应检测循环核糖核酸 ROS1 和 RET 融合记录的血液检测方法

从血液中检测循环核糖核酸 (茨尔纳) 是临床诊断中未满足的需要。在这里, 我们描述的方法, 茨尔纳的特点, 从非小细胞肺癌患者使用敏感和特定的数字聚合酶链反应。测试满足设计要求, 在72小时内检测融合变体。

We have developed novel methods for the isolation and characterization of tumor-derived circulating ribonucleic acid (cRNA) for blood-based liquid biopsy. ...

a-blood-based-test-for-detection-ros1-ret-fusion-transcripts-from

Nanyang Technological University

Research

JoVE Journal

Medicine

10.3K 次观看.  Biodesix. 从血液中检测循环核糖核酸 (茨尔纳) 是临床诊断中未满足的需要。在这里, 我们描述的方法, 茨尔纳的特点, 从非小细胞肺癌患者使用敏感和特定的数字聚合酶链反应。测试满足设计要求, 在72小时内检测融合变体。

视频: A Blood-based Test for the Detection of ROS1 and RET Fusion Transcripts from Circulating Ribonucleic Acid Using Digital Polymerase Chain Reaction

The Trier Social Stress Test Protocol for Inducing Psychological Stress

This article demonstrates a psychological stress protocol for use in a laboratory setting. Protocols that allow researchers to study the biological pathways of the stress response in health and disease are fundamental to the progress of research in stress and anxiety.1 Although numerous protocols exist for inducing stress response in the laboratory, many neglect to provide a naturalistic context or to incorporate aspects of social and psychological stress. Of psychological stress protocols, meta-analysis suggests that the Trier Social Stress Test (TSST) is the most useful and appropriate standardized protocol for studies of stress hormone reactivity.2 In the original description of the TSST, researchers sought to design and evaluate a procedure capable of inducing a reliable stress response in the majority of healthy volunteers.3 These researchers found elevations in heart rate, blood pressure and several endocrine stress markers in response to the TSST (a psychological stressor) compared to a saline injection (a physical stressor).3 Although the TSST has been modified to meet the needs of various research groups, it generally consists of a waiting period upon arrival, anticipatory speech preparation, speech performance, and verbal arithmetic performance periods, followed by one or more recovery periods. The TSST requires participants to prepare and deliver a speech, and verbally respond to a challenging arithmetic problem in the presence of a socially evaluative audience.3 Social evaluation and uncontrollability have been identified as key components of stress induction by the TSST.4 In use for over a decade, the goal of the TSST is to systematically induce a stress response in order to measure differences in reactivity, anxiety and activation of the hypothalamic-pituitary-adrenal (HPA) or sympathetic-adrenal-medullary (SAM) axis during the task.1 Researchers generally assess changes in self-reported anxiety, physiological measures (e.g. heart rate), and/or neuroendocrine indices (e.g. the stress hormone cortisol) in response to the TSST. Many investigators have adopted salivary sampling for stress markers such as cortisol and alpha-amylase (a marker of autonomic nervous system activation) as an alternative to blood sampling to reduce the confounding stress of blood-collection techniques. In addition to changes experienced by an individual completing the TSST, researchers can compare changes between different treatment groups (e.g. clinical versus healthy control samples) or the effectiveness of stress-reducing interventions.1

This article describes a protocol for inducing psychological stress in participants, which enables researchers to measure psychological, physiological and neuroendocrine responses to stress within single participants or between groups.

特里尔社会压力诱发心理应激的测试协议

This article demonstrates a psychological stress protocol for use in a laboratory setting.  Protocols that allow researchers to study the biological ...

科研

医学

The &alpha;-test: Rapid Cell-free CD4 Enumeration Using Whole Saliva

There is an urgent need for affordable CD4 enumeration to monitor HIV disease. CD4 enumeration is out of reach in resource-limited regions due to the time and temperature restrictions, technical sophistication, and cost of reagents, in particular monoclonal antibodies to measure CD4 on blood cells, the only currently acceptable method. A commonly used cost-saving and time-saving laboratory strategy is to calculate, rather than measure certain blood values. For example, LDL levels are calculated using the measured levels of total cholesterol, HDL, and triglycerides1. Thus, identification of cell-free correlates that directly regulate the number of CD4+ T cells could provide an accurate method for calculating CD4 counts due to the physiological relevance of the correlates.
The number of stem cells that enter blood and are destined to become circulating CD4+ T cells is determined by the chemokine CXCL12 and its receptor CXCR4 due to their influence on locomotion2. The process of stem cell locomotion into blood is additionally regulated by cell surface human leukocyte elastase (HLECS) and the HLECS-reactive active &alpha;1proteinase inhibitor (&alpha;1PI, &alpha;1antitrypsin, SerpinA1)3. In HIV-1 disease, &alpha;1PI is inactivated due to disease processes 4. In the early asymptomatic categories of HIV-1 disease, active &alpha;1PI was found to be below normal in 100% of untreated HIV-1 patients (median=12 &mu;M, and to achieve normal levels during the symptomatic categories4, 5. This pattern has been attributed to immune inactivation, not to insufficient synthesis, proteolytic inactivation, or oxygenation. We observed that in HIV-1 subjects with &gt;220 CD4 cells/&mu;l, CD4 counts were correlated with serum levels of active &alpha;1PI (r2=0.93, p&lt;0.0001, n=26) and inactive &alpha;1PI (r2=0.91, p&lt;0.0001, n=26) 5. Administration of &alpha;1PI to HIV-1 infected and uninfected subjects resulted in dramatic increases in CD4 counts suggesting &alpha;1PI participates in regulating the number of CD4+ T cells in blood 3.
With stimulation, whole saliva contains sufficient serous exudate (plasma containing proteinaceous material that passes through blood vessel walls into saliva) to allow measurement of active &alpha;1PI and the correlation of this measurement is evidence that it is an accurate method for calculating CD4 counts. Briefly, sialogogues such as chewing gum or citric acid stimulate the exudation of serum into whole mouth saliva. After stimulating serum exudation, the activity of serum &alpha;1PI in saliva is measured by its capacity to inhibit elastase activity. Porcine pancreatic elastase (PPE) is a readily available inexpensive source of elastase. PPE binds to &alpha;1PI forming a one-to-one complex that prevents PPE from cleaving its specific substrates, one of which is the colorimetric peptide, succinyl-L-Ala-L-Ala-L-Ala-p-nitroanilide (SA3NA). Incubating saliva with a saturating concentration of PPE for 10 min at room temperature allows the binding of PPE to all the active &alpha;1PI in saliva. The resulting inhibition of PPE by active &alpha;1PI can be measured by adding the PPE substrate SA3NA. (Figure 1). Although CD4 counts are measured in terms of blood volume (CD4 cells/&mu;l), the concentration of &alpha;1PI in saliva is related to the concentration of serum in saliva, not to volume of saliva since volume can vary considerably during the day and person to person6. However, virtually all the protein in saliva is due to serum content, and the protein content of saliva is measurable7. Thus, active &alpha;1PI in saliva is calculated as a ratio to saliva protein content and is termed the &alpha;1PI Index. Results presented herein demonstrate that the &alpha;1PI Index provides an accurate and precise physiologic method for calculating CD4 counts.

The &#945;-test: Rapid Cell-free CD4 Enumeration Using Whole Saliva

A CD4 enumeration method, the &#x03B1;-test, is described which uses whole saliva to provide rapid and accurate CD4 counts. The &alpha;-test costs pennies and eliminates the need for technical training, costly reagents such as monoclonal antibodies, instrumentation, refrigeration, transport of samples, as well as collection and handling of blood.

测试:α-细胞快速免费的CD4计数，使用全唾液

There is an urgent need for affordable CD4 enumeration to monitor HIV disease. CD4 enumeration is out of reach in resource-limited regions due to the time ...

Intraoperative Detection of Subtle Endometriosis: A Novel Paradigm for Detection and Treatment of Pelvic Pain Associated with the Loss of Peritoneal Integrity

Endometriosis is a common disease affecting 40 to 70% of reproductive-aged women with chronic pelvic pain (CPP) and/or infertility. The purpose of this study was to demonstrate the use of a blue dye (methylene blue) to stain peritoneal surfaces during laparoscopy (L/S) to detect the loss of peritoneal integrity in patients with pelvic pain and suspected endometriosis. Forty women with CPP and 5 women without pain were evaluated in this pilot study. During L/S, concentrated dye was sprayed onto peritoneal surfaces, then aspirated and rinsed with Lactated Ringers solution. Areas of localized dye uptake were evaluated for the presence of visible endometriotic lesions. Areas of intense peritoneal staining were resected and some fixed in 2.5% buffered gluteraldehyde and examined by scanning (SEM) electron microscopy. Blue dye uptake was more common in women with endometriosis and chronic pelvic pain than controls (85% vs. 40%). Resection of the blue stained areas revealed endometriosis by SEM and loss of peritoneal cell-cell contact compared to normal, non-staining peritoneum. Affected peritoneum was associated with visible endometriotic implants in most but not all patients. Subjective pain relief was reported in 80% of subjects. Based on scanning electron microscopy, we conclude that endometrial cells extend well beyond visible implants of endometriosis and appear to disrupt the underlying mesothelium. Subtle lesions of endometriosis could therefore cause pelvic pain by disruption of peritoneal integrity, allowing menstrual or ovulatory blood and associated pain factors access to underlying sensory nerves. Complete resection of affected peritoneum may provide a better long-term treatment for endometriosis and CPP. This simple technique appears to improve detection of subtle or near invisible endometriosis in women with CPP and minimal visual findings at L/S and may serve to elevate diagnostic accuracy for endometriosis at laparoscopy.

Loss of peritoneal integrity provides a new paradigm to understand and treat chronic pelvic pain in women with mild forms of endometriosis and can be easily detected using intraoperative instillation of dye at the time of laparoscopy.

术中的微妙的子宫内膜异位症的检测:A新型范式为相关联的与的的腹膜的完整性之亏损盆腔疼痛的的的的检测和治疗的

Endometriosis is a  common disease affecting 40 to 70% of reproductive-aged women with chronic  pelvic pain (CPP) and/or infertility. The purpose of this ...

Cerenkov Luminescence Imaging (CLI) for Cancer Therapy Monitoring

In molecular imaging, positron emission tomography (PET) and optical imaging (OI) are two of the most important and thus most widely used modalities1-3. PET is characterized by its excellent sensitivity and quantification ability while OI is notable for non-radiation, relative low cost, short scanning time, high throughput, and wide availability to basic researchers. However, both modalities have their shortcomings as well. PET suffers from poor spatial resolution and high cost, while OI is mostly limited to preclinical applications because of its limited tissue penetration along with prominent scattering optical signals through the thickness of living tissues.
 Recently a bridge between PET and OI has emerged with the discovery of Cerenkov Luminescence Imaging (CLI)4-6. CLI is a new imaging modality that harnesses Cerenkov Radiation (CR) to image radionuclides with OI instruments. Russian Nobel laureate Alekseyevich Cerenkov and his colleagues originally discovered CR in 1934. It is a form of electromagnetic radiation emitted when a charged particle travels at a superluminal speed in a dielectric medium7,8. The charged particle, whether positron or electron, perturbs the electromagnetic field of the medium by displacing the electrons in its atoms. After passing of the disruption photons are emitted as the displaced electrons return to the ground state. For instance, one 18F decay was estimated to produce an average of 3 photons in water5. 
 Since its emergence, CLI has been investigated for its use in a variety of preclinical applications including in vivo tumor imaging, reporter gene imaging, radiotracer development, multimodality imaging, among others4,5,9,10,11. The most important reason why CLI has enjoyed much success so far is that this new technology takes advantage of the low cost and wide availability of OI to image radionuclides, which used to be imaged only by more expensive and less available nuclear imaging modalities such as PET.
 Here, we present the method of using CLI to monitor cancer drug therapy. Our group has recently investigated this new application and validated its feasibility by a proof-of-concept study12. We demonstrated that CLI and PET exhibited excellent correlations across different tumor xenografts and imaging probes. This is consistent with the overarching principle of CR that CLI essentially visualizes the same radionuclides as PET. We selected Bevacizumab (Avastin; Genentech/Roche) as our therapeutic agent because it is a well-known angiogenesis inhibitor13,14. Maturation of this technology in the near future can be envisioned to have a significant impact on preclinical drug development, screening, as well as therapy monitoring of patients receiving treatments.

Cerenkov Luminescence Imaging CLI for Cancer Therapy Monitoring

Use of Cerenkov Luminescence Imaging (CLI) for monitoring preclinical cancer treatment is described here. This method takes advantage of Cerenkov Radiation (CR) and optical imaging (OI) to visualize radiolabeled probes and thus provides an alternative to PET in preclinical therapeutic monitoring and drug screening.

癌症治疗监测的切伦科夫发光成像（CLI）

In  molecular imaging, positron emission tomography (PET) and optical imaging (OI)  are two of the most important and thus most widely used modalities1-3. ...

Assessment of Gastric Emptying in Non-obese Diabetic Mice Using a [13C]-octanoic Acid Breath Test

Gastric emptying studies in mice have been limited by the inability to follow gastric emptying changes in the same animal since the most commonly used techniques require killing of the animals and postmortem recovery of the meal1,2. This approach prevents longitudinal studies to determine changes in gastric emptying with age and progression of disease. The commonly used [13C]-octanoic acid breath test for humans3 has been modified for use in mice4-6 and rats7 and we previously showed that this test is reliable and responsive to changes in gastric emptying in response to drugs and during diabetic disease progression8. In this video presentation the principle and practical implementation of this modified test is explained. As in the previous study, NOD LtJ mice are used, a model of type 1 diabetes9. A proportion of these mice develop the symptoms of gastroparesis, a complication of diabetes characterized by delayed gastric emptying without mechanical obstruction of the stomach10.
This paper demonstrates how to train the mice for testing, how to prepare the test meal and obtain 4 hr gastric emptying data and how to analyze the obtained data. The carbon isotope analyzer used in the present study is suitable for the automatic sampling of the air samples from up to 12 mice at the same time. This technique allows the longitudinal follow-up of gastric emptying from larger groups of mice with diabetes or other long-standing diseases.

Assessment of Gastric Emptying in Non-obese Diabetic Mice Using a [13C]-octanoic Acid Breath Test

Determination of gastric emptying with a non-invasive [13C]-octanoic acid breath test for tracking gastroparesis in female NOD LtJ mice.

使用的非肥胖型糖尿病小鼠胃排空的评估[<sup&gt; 13</sup&gt; C-辛酸呼气试验

Gastric emptying studies in mice have been limited by the inability to follow gastric emptying changes in the same animal since the most commonly used ...

Isolation and Propagation of Circulating Tumor Cells from a Mouse Cancer Model

Cancer metastasis is the foremost cause of cancer-associated deaths. Recent studies have shown that circulating tumor cells (CTCs) are important in cancer metastasis. Indeed, the number of CTCs correlates with tumor size. Here, a detailed description is provided of a methodology for isolation and propagation of CTCs from a syngeneic mouse model of hepatocellular carcinoma (HCC) which allows for downstream analysis of potentially important molecular mechanisms of solid organ tumor metastasis. This method is efficient and reproducible. It is a non-invasive technique and, therefore, has potential to replace the invasive biopsy of tissues from humans which may be associated with complications. Therefore, the method discussed here allows for the isolation and propagation of CTCs from whole blood samples such that they can be examined and characterized. This has potential for future adaptation for clinical applications such as diagnosis, and personalized targeted therapy.

Circulating tumor cells (CTCs) have been shown to play an important role in tumor metastasis. Here, a method for the isolation and propagation of CTCs from the whole blood of a syngeneic mouse tumor model of hepatocellular carcinoma (HCC) metastasis is described.

从小鼠癌症模型的循环肿瘤细胞的分离和繁殖

Cancer metastasis is the foremost cause of cancer-associated deaths. Recent studies have shown that circulating tumor cells (CTCs) are important in cancer ...

Novel Diagnostics in Revision Arthroplasty: Implant Sonication and Multiplex Polymerase Chain Reaction

In orthopedic patients, foreign body-associated infections, especially periprosthetic joint infections (PJIs), are a devastating complication of arthroplasty. Infection requires complex treatment, may result in long hospitalization and causes considerable costs. Multiple surgical revisions can be necessary in these patients, with a loss in function as well as in quality of life.
The routine preoperative diagnostics include blood examination for C-reactive protein (CRP) and other biomarkers, as well as joint aspirate analysis for cell count, differentiation, and culture. Intraoperative specimens for histology and microbiology are also standard procedure. The microbiological examination of removed implants with sonication, in combination with the implementation of molecular biology techniques in microbiology, represent two novel techniques currently employed to enhance the differential diagnostics of PJI.
We present here the step-wise procedure of analyzing joint aspirate and sonication fluid, using a cartridge-based multiplex polymerase chain reaction (PCR) system. Results were matched against conventional cultures and consensus criteria for PJI. Conventional microbiological cultures from tissue biopsies, joint aspirate and sonication fluid showed a sensitivity of 66.7%, 66.7%, and 88.9%, respectively, and a specificity of 82.3%, 54.6%, and 61.5%, respectively. The PCR diagnostic of the sonication fluid and the joint fluid showed a sensitivity of 50.0% and 55.6%, respectively, and both a specificity of 100.0%. Both PCR diagnostics combined had a sensitivity of 66.7% and a specificity of 100.0%. The multiplex PCR therefore presents a rapid diagnostic tool with moderate sensitivity but high specificity in diagnosing PJI.

Differential diagnostics in painful arthroplasty is crucial for treatment success. Joint aspiration is routinely performed preoperatively. Multiplex polymerase chain reaction of the joint aspirate and the sonication fluid, a feasible tool for quick pathogen detection from these samples is described.

翻修术中的新诊断: 种植超声和多重聚合酶链反应

In orthopedic patients, foreign body-associated infections, especially periprosthetic joint infections (PJIs), are a devastating complication of ...

Taste Exam: A Brief and Validated Test

The emerging importance of taste in medicine and biomedical research, and new knowledge about its genetic underpinnings, has motivated us to supplement classic taste-testing methods in two ways. First, we explain how to do a brief assessment of the mouth, including the tongue, to ensure that taste papillae are present and to note evidence of relevant disease. Second, we draw on genetics to validate taste test data by comparing reports of perceived bitterness intensity and inborn receptor genotypes. Discordance between objective measures of genotype and subjective reports of taste experience can identify data collection errors, distracted subjects or those who have not understood or followed instructions. Our expectation is that fast and valid taste tests may persuade researchers and clinicians to assess taste regularly, making taste testing as common as testing for hearing and vision. Finally, because many tissues of the body express taste receptors, taste responses may provide a proxy for tissue sensitivity elsewhere in the body and, thereby, serve as a rapid, point-of-care test to guide diagnosis and a research tool to evaluate taste receptor protein function.

This protocol measures human taste responses and includes a brief anatomical assessment, a short taste test, and a validation method using the subject&#39;s reported sensation and taste receptor genotype.

味觉考试: 一个简短的验证测试

The emerging importance of taste in medicine and biomedical research, and new knowledge about its genetic underpinnings, has motivated us to supplement ...

Digital PCR for Quantifying Circulating MicroRNAs in Acute Myocardial Infarction and Cardiovascular Disease

Circulating serum microRNAs (miRNAs) have shown promise as biomarkers for the cardiovascular disease and acute myocardial infarction (AMI), being released from the cardiovascular cells into the circulation. Circulating miRNAs are highly stable and can be quantified. The quantitative expression of specific miRNAs can be linked to the pathology, and some miRNAs show high tissue and disease specificity. Finding novel biomarkers for cardiovascular diseases is of importance for medical research. Quite recently, digital polymerase chain reaction (dPCR) has been invented. dPCR, combined with fluorescent hydrolysis probes, enables specific direct absolute quantification. dPCR exhibits superior technical qualities, including a low variability, high linearity, and high sensitivity compared to the quantitative polymerase chain reaction (qPCR). Thus, dPCR is a more accurate and reproducible method for directly quantifying miRNAs, particularly for the use in large multi-center cardiovascular clinical trials. In this publication, we describe how to effectively perform digital PCR in order to assess the absolute copy number in serum samples.

Circulating microRNAs have shown promise as biomarkers for cardiovascular diseases and acute myocardial infarctions. In this study, we describe a protocol for miRNA extraction, reverse transcription, and digital PCR for the absolute quantification of miRNAs in the serum of patients with cardiovascular disease.

数字 PCR 在急性心肌梗死和心血管疾病中量化循环 MicroRNAs 的临床观察

Circulating serum microRNAs (miRNAs) have shown promise as biomarkers for the cardiovascular disease and acute myocardial infarction (AMI), being released ...

Traditional Trail Making Test Modified into Brand-new Assessment Tools: Digital and Walking Trail Making Test

The Trail Making Test (TMT) is a well-accepted tool for evaluating executive function. The standard TMT was invented more than 60 years ago and has been modified into many versions. With the development of digital technologies, TMT is now modified to a digitized version. The present study demonstrated digital TMT (dTMT) performed on a computer, and Walking TMT (WTMT) on the floor. Both revealed more information compared with the traditional version of TMT.

Here, we present a protocol to show how to perform two types of cognitive assessment tools derived from the paper-pencil version of the Trail Making Test.

传统跟踪测试修改为全新的评估工具:数字和步行路线制作测试

The Trail Making Test (TMT) is a well-accepted tool for evaluating executive function. The standard TMT was invented more than 60 years ago and has been ...