这项工作得到 (1) 支持的乳腺癌项目奖学金 (2014-2016) 资助的行动为乳腺癌基金会和活2013通过研究, 创新 & 发展信托 (RIDT) 的马耳他大学, (2) 医学学院& 外科, 马耳他大学和 (3) 由马耳他科学 & 技术理事会通过融合资助的项目法案: R & I 技术开发方案2016。这篇手稿的出版是通过朱庇特-Luminex 赠款来支持的。

从马耳他大学研究伦理学委员会 (UREC) 获得了在本研究中使用乳腺组织材料的伦理批准 (Ref: 22/2012)。
1. 组织准备
<ol><li>使用适当的 H & E 程序14, 准备染色的参考组织部分和数字扫描幻灯片, 以供参考在显微切割。</li>
	<li>使用切片, 减少20µm 组织部分到一个 RNAse 无水浴在40°c。用清洁的、RNase 的设备和材料收集薄膜片上的激光显微切片。</li>
	<li>干燥的膜在干燥孵化室过夜37°c。染色采用适当的 H & E 程序15与分子生物学级溶液和增加染色时间的苏木精到6分钟。如果需要, 用适当的免疫组化协议16染色膜的幻灯片。</li>
</ol>2. 激光显微切割
<ol><li>设置幻灯片限制并校准舞台运动。<ol>
<li>将激光视图移动到薄膜幻灯片的空白区域。通过在增加焦距间隔 (5%) 的情况下发射激光镜头, 校准激光聚焦, 直到激光开始穿透膜。绘制和 microdissect 任何形状和微调激光聚焦, 以最大限度地提高激光切割过程中的激光器效率。</li>
		<li>将舞台运动速度提高到激光剥离效率不受损的地步。校准激光在选定的物镜和重新校准, 如果改变目标 (激光速度在 1–15%, 重点在 70–75%, 并在90–100% 时, 使用4X 目标)。</li>
	</ol>
</li>
	<li>使用4X 目标扫描彩色膜幻灯片。</li>
	<li>选择并环绕在膜片上进行显微切割的目标区域。激光解剖最小面积42毫米2。记录所解剖的区域, 以确定要使用的样本均匀化缓冲区的体积。</li>
	<li>使用帽升降机构将解剖部分检索到附加在相应附属物上的标记管的扩散器盖上。如果未将解剖剖面检索到盖上, 请重复该区域的激光解剖。</li>
</ol>3. 组织裂解
注: 一些解决方案和材料连同材料表中提到的套件一起提供。
<ol><li>用均匀溶液和蛋白酶 K 在60:1 的比例下制备所需的均质混合物体积以进行样品裂解。</li>
	<li>吸管2.4 µL 均匀溶液入每个管为每1毫米2的组织面积, 漩涡为十年代以最大速度, 并且离心机在室温下为 5 s 在 2500 x g。</li>
	<li>在加热块中孵育, 在65摄氏度的 12–18 h 与震动在 600 rpm。</li>
	<li>在室温下离心裂解物 2.1万 x 克, 5 分钟。</li>
	<li>使用吸管, 小心地吸清上清, 并分配到一个标签新鲜的管。避免吸出组织/膜碎片, 因为这可能会降低结果的质量。</li>
	<li>在-80 摄氏度冷藏库中储存清上清液。</li>
</ol>4. 基于杂交的化验
<ol><li>执行下列准备工作。<ol>
<li>将试剂瓶放置在37°c 30 分钟的孵化器中, 并通过反转轻轻混合, 预热裂解混合物。</li>
		<li>如果组织组织匀浆被冷冻, 室温下解冻, 在37摄氏度孵育30分钟. 在孵化后的最大速度下, 将样品涡流。</li>
		<li>将震动孵化器设置为54摄氏度, 将温度验证套件的探头放置在96井板的空井中。2小时后, 检查温度的数字温度计和设置的三角洲温度的震动孵化器, 以纠正任何温差。</li>
		<li>解冻和漩涡的探针集和阻断试剂十年代, 离心机在室温下为5秒在 2500 x g, 并保持冰。</li>
		<li>使用时, 把蛋白酶 K 瓶放在冰上。</li>
		<li>油脂实验捕获珠在46赫为3分钟, 然后漩涡在最大速度为另3分钟。</li>
	</ol>
</li>
	<li>通过扩大以下试剂, 准备25µL 工作珠混合: 4.25 µL 的核酸酶水, 16.65 µL 的裂解混合物, 1.00 µL 的阻断试剂, 0.10 µL 的蛋白酶 K, 0.50 µL 捕获珠, 2.50 µL 的探针集。</li>
	<li>旋涡的工作珠混合在十年代的最大速度, 然后吸管25µL/井进入磁分离板。</li>
	<li>吸管25µL 组织匀浆, 以磁分离96井板和25µL 均质溶液为3井作为空白。</li>
	<li>使用明确的塑料压力密封密封磁选板, 并将该板放在孵化器的 54, 1 °c 为 18–22 h, 而颤抖在 600 rpm。</li>
	<li>通过将31.5 毫升的 RNase 水与0.1 毫升的洗涤缓冲元件1和1.67 毫升的洗涤缓冲元件2混合, 为24口井准备1X 洗涤缓冲液。</li>
	<li>从孵化器中取出盘子。将震动孵化器的温度设置为50至1摄氏度。</li>
	<li>将磁选板安装在手持式磁板垫圈上, 并锁定到位。卸下压力密封, 等待1分钟的磁珠沉淀。</li>
	<li>洗涤步骤: 将磁选板倒置在水槽上, 轻轻涂抹在纸巾上, 以去除残余溶液。使用多通道吸管在每个井中添加100µL 的1X 洗涤缓冲液, 并等待十五年代. 重复这一步, 洗盘子3次。</li>
	<li>吸管50µL 的前放大器试剂对每个井。用铝板密封密封板, 在室温下用 800 rpm 将板材摇动1分钟。孵育1小时在 50, 1 °c, 而颤抖在 600 rpm。</li>
	<li>重复步骤 4.9 (洗涤步骤)。</li>
	<li>吸管50µL 的放大器试剂对每个井。用铝板密封密封板, 在步骤4.10 中摇动板材。</li>
	<li>重复步骤 4.9 (洗涤步骤)。</li>
	<li>漩涡标签探针十年代以最大速度。将标签探头的50µL 添加到每个井中。密封和摇动板 (步骤 4.10)。</li>
	<li>重复步骤 4.9 (洗涤步骤)。</li>
	<li>涡旋的链亲和素藻红蛋白 (SAPE) 在十年代的最大速度。每井加50µL SAPE。密封和摇动板 (步骤 4.10)。</li>
	<li>重复步骤 4.9 (洗涤步骤), 除了使用 SAPE 洗涤缓冲器, 而不是洗涤缓冲液。</li>
	<li>添加130µL 的 SAPE 洗涤缓冲器到每个井和密封板与铝板密封。在室温下以800转每分钟摇动盘子3分钟。</li>
	<li>启动磁珠分析仪。按照制造商的指示执行校准和验证程序。</li>
	<li>使用以下参数在磁珠分析仪上建立协议: 样品尺寸: 100 µL;DD 门: 5,000–25,000;超时时间: 九十年代;珠状活动/珠区: 100;然后单击 "下一步"。在面板中选择相应的珠号, 并根据制造商的指示, 定义珠区域和相应的目标基因。</li>
	<li>通过从 "批次" 选项卡中选择 "使用现有协议创建批处理" 来添加新分析。在板块布局中, 分别指定井为未知或空白, 并为 "样品" 面板中的每个样品提供一个标签。</li>
	<li>从96井反应板上取下铝板密封件。通过点击弹出按钮弹出盘子托盘, 将板块加载到磁珠分析仪中。单击退刀|运行批处理以启动分析器。阅读后, 弹出并卸下96井板。根据制造商的建议, 清洁并关闭磁珠分析仪。</li>
</ol>5. 数据分析
<ol><li>在批处理|结果"选项卡上, 选择相应的分析文件, 然后单击"导出到. csv "按钮。使用电子表格软件打开. csv 文件。</li>
	<li>观察珠数和省略任何结果 < 40 珠/地区。</li>
	<li>平均出空白井的值, 并计算出标准偏差 (SD) 和检测限度 (LOD) (空白平均 + (3xSD)) 每个目标基因。将低于 LOD 的值设置为未检测。 注意: 如果任何正则表达式值未被检测到, 则认为示例数据不充分。考虑表达式值超过2万以上的灵敏度上限。这些样品的裂解物应稀释和重新分析。</li>
	<li>减去每个基因的原始数据的空白平均值。</li>
	<li>计算每个样本所选规范化基因的几何平均值。将单个示例数据规范化为相应的几何平均值。</li>
	<li>分析数据科学平台上的数据或使用定义的算法。<ol>
<li>通过单击 "添加数据" 将数据导入到数据科学平台中。将数据文件拖到流程工作区中, 然后连接到 [选择属性]运算符, 然后再转到主组件分析 (PCA)运算符, 然后再转到结果t 端口。</li>
		<li>在选择属性运算符中, 选择规范化的基因数据的子集和一个名词性变量, 如乳腺癌亚型。单击 "播放" 运行该进程。在 "图表" 选项卡中, 选择图表样式中的 "散射3D 颜色" 和 "颜色" 字段中的标称变量。在3维图上评估 PCA 数据的变异性。</li>
	</ol>
</li>
</ol>

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Invitrogen™ QuantiGene™丛试验是热费希尔科学的专有。

通过对 FFPE 乳腺癌组织和正常乳腺导管的降解 RNA 的基因表达进行优化, 建立了一种基于珠子的复 bDNA 检测方法。优化检测, 涉及开发一种方法, 以分类乳腺癌肿瘤的腔内和基底亚型利用8著名的生物标志物和5个潜在的规范化基因。利用规范化基因的置换进行数据规范化。规范化基因的选择是基于对受体状态的最佳预测, 采用腔内/基底分类器基因。为了对 FFPE 组织内腔/基底亚型进行分类, 选择的规范化基因为β肌动蛋白 (ACTB), 甘油 3-磷酸脱氢酶 (GAPDH), 嘌呤 Phosphoribosyltransferase 1 (HPRT1)。
该方法可用于其他诊断和研究领域, 在适当选择规范化的基因集后。该方法在研究部门中的一个重要应用是对档案材料中生物标志物的测量, 并对临床结果进行了充分的标注。这可以快速准确地验证回顾性研究中潜在的预测标记, 并避免长期前瞻性研究, 等待无疾病生存和整体生存数据。目前, 我们的小组正在调查使用的检测, 以发现受体阳性外来体, 这需要开发一个新的算法, 利用交替规范化基因的数据规范化。使用液体活检和强健的基因表达检测, 将允许高通量复用检测在治疗期间适应患者管理, 并提供一种方法, 以遵循治疗效果, 潜在复发由于抗治疗, 以及肿瘤的转移能力。
该方法在肿瘤诊断中也有广泛的应用, 并适应目前的诊断工作流程。该方法在诊断领域的主要优点包括: (1) 高通量检测的实施, (2) 不包括基于图像测量的主观性和模棱两可的结果 (3) 对多个目标的精确探测同时, 这提高了准确性和尽量减少宝贵的病人样本的使用, (4) 不需要高度专业化的设施和人力资源。优化的取样过程, 连同珠基复用试验所需材料的低输入, 可以进一步研究肿瘤的异质性;通过激光显微切割, 准确地分离出同一患者部分恶性组织的多个病灶, 可以比较它们之间的多种基因表达以及与正常组织的匹配 (图 4)。低材料输入对肿瘤活检提供有限的肿瘤组织的诊断应用至关重要。通过测定降解 RNA 样品中基因表达的能力, 可以方便地在机构内进行分析, 或为实验室提供服务。此外, 还可以使用 H & E 染色材料进行全断面分析 (图 1)。
为使本议定书的成功, 必须: (1) 确保正确抽样的肿瘤现场/裂解的检测和 (2) 开发良好的优化和验证数据规范化算法, 每个基因表达板和/或个人预后或预测性生物标志物。前者取决于执行取样的技术员/科学家的技术经验。建议采取一个额外的核心, 并准备一个组织微阵列 (TMA) 相同的格式的复合磁珠检测 (96 井格式)。这将提供一个肿瘤网站的档案作为一个副本的样本用于 RNA 的化验。TMAs 还可以用其他技术进行评估, 以进行后续研究或验证结果。规范化算法的发展依赖于所研究的材料和规范化的规范化基因。根据样品中表达的水平和变异性, 选择不同的规范化基因组, 不同来源的癌组织、血浆外来体或循环肿瘤细胞之间存在差异。试验验证包括样品处理, 因为各种制剂也会产生不同的规范化算法。
综上所述, 利用 bDNA 技术与磁珠技术结合, 选择合适的靶基因组, 将提供直接在少量的组织裂解物中测量基因表达的额外优势。患者材料, 包括 microdissected 材料、外来体和循环肿瘤细胞。除了检测肿瘤的异质性, 适当使用面板有可能检测肿瘤衍生外来体早期诊断和早期发现复发。由于不需要核酸扩增步骤, 采用 bDNA 技术的信号放大, 结合珠基复用, 在临床说明的档案材料中测量多种基因表达, 为生物标志物验证。

利用档案福尔马林固定石蜡 (FFPE) 材料进行 rna 检测的优化研究由于组织完整性保存1的福尔马林所引起的手术组织处理和 RNA 降解的变化而具有挑战性, 2。为了克服从档案材料中进行准确基因表达研究的局限性, 本组采用了 bDNA 复合磁珠法。bDNA 技术不采用酶法放大目标模板, 而是使用特定探针的杂交和放大3的报告信号。捕获和检测探针的短识别序列旨在杂交目标 RNA4的短片段。此外, 使用组织组织匀浆作为直接启动材料的这一化验, 克服了不可避免的损失 rna 的结果, 由化验需要事先 RNA 提取和纯化。信号放大, 使用短识别序列, 排除纯化步骤, 有助于降低检测技术的变化。该技术提供了多路检测 (最多80个 RNA 目标) 的可能性, 并测量了低材料输入下的目标面板的表达式。本协议描述了激光显微切割组织样品的制备和染色。细胞膜上的染色有助于肿瘤和组织学结构的成像, 提供准确的选择和分析: (1) 乳腺组织中的肿瘤和正常导管, 以及 (2) 异质肿瘤内的恶性细胞克隆。
乳腺癌的分子分类是一种将患者肿瘤归类为三个分子类的过程,即腔内、人表皮生长因子受体 2 (HER2) 富集和基底亚型。HER2-enriched 亚型具有良好的定义, HER2 受体的表达高, ERBB2 基因放大, 结合低或无雌激素受体 (ER) 和孕激素受体 (PgR)。腔内亚型一般为 ER 阳性, 基底亚型一般为三受体 (HER2, ER, PgR) 阴性, 与三重阴性乳腺癌 (TNBC) 诊断亚型5,6有显著的重叠。其他标记用于确定上皮和间充质特征。纤维连接蛋白 (FN1) 是乳腺组织间充质室的主要成分。增加的 FN1 表达伴随着高 Ki67 染色, 并显示一个更具侵袭性的肿瘤7,8和与转移9相关的签名。有趣的是, FN1 被发现是存在于微泡起源于肿瘤细胞, 这诱导激活有丝分裂信号在受体成纤维细胞10。因此, 循环微泡, 如外来体是一个潜在的标志, 早期发现或转移和复发11。
乳腺癌转录子类型) 的肿瘤面积选择反复由 macrodissection12、13进行。为了克服组织异质性和提高灵敏度, 我们有可靠地结合经典组织染色与多重分子分析方法。两种不同的乳腺癌无性系作为原则的证明, 其上皮间质特征和转移电位均有明确的定义。描述的协议的工作流可以很容易地转化为目前的临床设置, 并用于选择性地分离和表征组织亚型使用目标 mRNA 分析。

<table><tbody><tr><td>Microtome</td><td>Leica</td><td>RM2235</td><td></td></tr><tr><td>Heamatoxylin Mayer&#039;s</td><td>Sigma</td><td>MHS16-500mL</td><td></td></tr><tr><td>Eosin Y Aquaeous solution</td><td>Sigma</td><td>HT110216-500mL</td><td></td></tr><tr><td>Normal Rabbit Serum</td><td>Monosan</td><td>MONX10963</td><td>Working dilution: 1/40</td></tr><tr><td>Biotinylated Rabbit anti-mouse</td><td>Dako</td><td>E0354</td><td>Working dilution: 1/200</td></tr><tr><td>ER antibody (6F11)</td><td>Vector Laboratories</td><td>VPE614</td><td>Working dilution: 1/45</td></tr><tr><td>HER2 antibody (CB11)</td><td>Novocastra</td><td>CB11-L-CE</td><td>Working dilution: 1/325</td></tr><tr><td>Ki67 antibody (MIB-1)</td><td>Dako</td><td>M7240</td><td>Working dilution: 1/500</td></tr><tr><td>Avidin Biotin Complex kit</td><td>Vector Laboratories</td><td>PK-6100</td><td></td></tr><tr><td>Nikon Eclipse Ti-E Inverted microscope</td><td>Nikon</td><td>Ti-E</td><td>4x, 10x, 20x and 40x objectives</td></tr><tr><td>Laser Microdissection membranes</td><td>Molecular Machines &amp;amp;Industries</td><td>S0103</td><td></td></tr><tr><td>mmi CellCamera 1.4</td><td>Molecular Machines &amp;amp;Industries</td><td>MX4285c-ACK07</td><td></td></tr><tr><td>mmi Cellcut Plus</td><td>Molecular Machines &amp;amp;Industries</td><td></td><td></td></tr><tr><td>Diffuser caps</td><td>Molecular Machines &amp;amp;Industries</td><td>50210</td><td></td></tr><tr><td>mmi Celltools Software v.4.01rcl</td><td>Molecular Machines &amp;amp;Industries</td><td></td><td></td></tr><tr><td>Eppendorf Thermomixer comfort</td><td>Eppendorf</td><td>5355000038</td><td></td></tr><tr><td>1.5mL heating block for Eppendorf Thermomixer</td><td>Eppendorf</td><td>22670522</td><td></td></tr><tr><td>96-well plate heating block for Eppendorf Thermomixer</td><td>Eppendorf</td><td>22670565</td><td></td></tr><tr><td>Labnet Vortemp 56 Shaking incubator</td><td>Labnet</td><td>52056A-220</td><td></td></tr><tr><td>LX200 100/200</td><td>Luminex</td><td></td><td>Magnetic bead analyser</td></tr><tr><td>Invitrogen QuantiGene Sample Processing Kit - FFPE Tissues</td><td>ThermoFisher Scientific</td><td>QS0109</td><td></td></tr><tr><td>Invitrogen QuantiGene Plex 2.0 Assay Kit (Magnetic Separation)</td><td>ThermoFisher Scientific</td><td>QP1011</td><td></td></tr><tr><td>Thermaseal RTS Sealing Film</td><td>Thermaseal</td><td>765246</td><td></td></tr><tr><td>Hand-Held Magnetic Plate Washer</td><td>ThermoFisher Scientific</td><td>QP1011</td><td></td></tr><tr><td>Invitrogen QuantiGene Incubator Temperature Validation Kit</td><td>Affymetrix/Panomics</td><td>QS0517</td><td></td></tr><tr><td>Proteinase K (50&amp;micro;g/&amp;micro;L)</td><td>ThermoFisher Scientific</td><td>14622</td><td></td></tr><tr><td>Invitrogen QuantiGene Plex 2.0 Sets</td><td>ThermoFisher Scientific</td><td>Various</td><td></td></tr><tr><td>Multi Speed Vortex</td><td>Kisker Biotech</td><td>MSV-3500</td><td></td></tr><tr><td>Sonicator</td><td>Silvercrest</td><td></td><td></td></tr><tr><td>RNASEZAP</td><td>Sigma</td><td>R2020-250ML</td><td></td></tr><tr><td>Aluminium 96-well plate seal</td><td>Sigma</td><td>Z721549-100EA</td><td></td></tr><tr><td>Temperature Validation Kit</td><td>ThermoFisher Scientific</td><td>QS0517</td><td></td></tr><tr><td>RapidMiner Studio Community 7.1.001</td><td>RapidMiner</td><td></td><td>Data Science Platform</td></tr><tr><td>Hybridisation oven</td><td>Hybaid (Thermo Scientific)</td><td></td><td></td></tr></tbody></table>

optimization of a multiplex rna-based expression assay using breast cancer archival material

核酸在档案组织中的降解, 肿瘤的异质性, 以及缺乏新鲜冰冻组织标本, 会对全球病理实验室的癌症诊断服务产生负面影响。基因放大和表达诊断测试使用档案材料或材料, 需要运输到服务实验室, 需要一个更可靠和准确的测试, 适应目前的临床工作流程。我们的研究小组优化了使用 Invitrogen™ QuantiGene™丛分析 (热费舍尔科学), 以量化 RNA 的档案材料使用分支 DNA (bDNA) 技术 Luminex xMAP®磁性珠。本论文描述的基因表达法是一种新颖、快速、多样的方法, 能够将乳腺癌准确地归类为不同的分子亚型, 省略了成像技术固有的解释主观性。另外, 由于所需材料的输入量低, 异质肿瘤可采用苏木精和 microdissected (H & E) 染色切片进行激光照射。该方法有广泛的可能应用, 包括肿瘤分类的诊断潜力和测量生物标志物在液体活检, 这将使更好的病人管理和疾病监测。此外, 对档案材料中生物标志物的定量测量在肿瘤学研究中是有用的, 可以利用临床标注材料的库, 追溯性研究能够验证潜在的生物标志物及其临床效果。相关。

所述方法已应用于 H & E 染色40个记录的同时测量 (图 1), microdissected (图 2) 高度退化的 FFPE 材料。利用该方法, 对肿瘤患者的受体状态 (图 3A)、肿瘤的分类和基底分子亚型17进行了准确的描述, 并 FN1 了间充质标记的差异表达。和配对控制组织 (图 3B), 在各种受体阳性和阴性亚型。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57148/57148fig1.jpg"> 图 1: 使用 H & E 染色材料进行基因表达.与染色的肿瘤切片相比, 从无瑕肿瘤切片获得的表达谱之间的相关性。[皮尔逊相关p值 = 5.34E-26]。转载于许可17。<a href="https://www.jove.com/files/ftp_upload/57148/57148fig1large.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/57148/57148fig2.jpg"> 图 2: FFPE 组织的激光显微切割.(A) & 彩色滑梯;(B) ER 表达的免疫组化染色;(C) HER2 免疫组化染色;(D) 无瑕20µm 的激光显微切割膜切片。黄色箭头表示有浸润性肿瘤的区域由于缺乏染色而未明确标定。(E) 与 H & E 染色的20µm 段, 以便更好地划定感兴趣的领域。白色箭头表示激光解剖轨迹, 而红色箭头显示激光聚焦在解剖过程中。所有插图均以10x 倍放大率拍摄。<a href="https://www.jove.com/files/ftp_upload/57148/57148fig2large.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/57148/57148fig3.jpg"> 图 3: (a) 受体状态的表达和 (B) 间充质标记 FN1, 在乳腺肿瘤中与正常对照.经典诊断乳腺癌亚型定义为每诊断结果使用免疫组化和荧光原位杂交 (鱼) HER2 模棱两可的免疫组化染色。HER2 阳性、er 阳性和 TNBC 病例与正常乳腺组织相比, 以杂交为基础测定的 (a) HER2 和 er (B) FN1 的规范化表达。克鲁斯卡尔沃利斯试验统计 (K W χ2) 表明, 肿瘤组织中 HER2 的表达显著 (p < 0.05) 高于 HER2 阳性队列中匹配的正常组织, TNBC 队列中显著降低。在肿瘤组织中, er 表达不明显高于 er 阳性和 TNBC 组的匹配正常。FN1 在 HER2 和 ER 阳性亚型中的肿瘤组织中显著增高, TNBC 组的肿瘤组织也呈显著升高趋势。<a href="https://www.jove.com/files/ftp_upload/57148/57148fig3large.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/57148/57148fig4.jpg"> 图 4: 病例研究: 肿瘤异质性.形态学上不同的肿瘤被 microdissected 并作为不同的标本进行治疗。(A) H & E 部分的主扫描。(B、 C)一个10x 和40x 的放大倍数, 分别为每个肿瘤形态学鉴定。(D) 以10x 倍的比例对 ER 进行免疫组化染色。(E) Ki67 的免疫组化染色, 显示肿瘤1的有丝分裂活性较高。(F) 每个肿瘤中 ESR1 基因的规范化表达水平, 显示肿瘤与免疫组化结果之间的相对较高和相等的表达。(G) 规范化表达水平的 FN1, 一个间充质标记, 增加 FN1 表达伴随着高 Ki67 染色显示一个更具侵袭性肿瘤的签名。在肿瘤2中观察到逆, 这似乎是一种较慢的增殖肿瘤, 其恶性电位降低 FN1 表达。<a href="https://www.jove.com/files/ftp_upload/57148/57148fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Optimization of a Multiplex RNA-based Expression Assay Using Breast Cancer Archival Material at JoVE.com

Optimization of a Multiplex RNA-based Expression Assay Using Breast Cancer Archival Material

核酸在档案组织中的降解, 肿瘤的异质性, 以及缺乏新鲜冰冻组织标本, 会对全球病理实验室的癌症诊断服务产生负面影响。这篇手稿描述了一组生物标志物的优化使用多重磁珠法分类乳腺癌。

应用乳腺癌档案材料的多路 RNA 表达方法的优化

Nucleic acid degradation in archival tissue, tumor heterogeneity, and a lack of fresh frozen tissue specimens can negatively impact cancer diagnostic ...

optimization-multiplex-rna-based-expression-assay-using-breast-cancer

Research

JoVE Journal

Bioengineering

7.8K 次观看.  University of Malta. 核酸在档案组织中的降解, 肿瘤的异质性, 以及缺乏新鲜冰冻组织标本, 会对全球病理实验室的癌症诊断服务产生负面影响。这篇手稿描述了一组生物标志物的优化使用多重磁珠法分类乳腺癌。

视频: Optimization of a Multiplex RNA-based Expression Assay Using Breast Cancer Archival Material

High Throughput MicroRNA Profiling: Optimized Multiplex qRT-PCR at Nanoliter Scale on the Fluidigm Dynamic ArrayTM IFCs

The broad involvement of miRNAs in critical processes underlying development, tissue homoeostasis and disease has led to a surging interest among the research and pharmaceutical communities. To study miRNAs, it is essential that the quantification of microRNA levels is accurate and robust. By comparing wild-type to small RNA deficient mouse embryonic stem cells (mESC), we revealed a lack of accuracy and robustness in previous published multiplex qRT-PCR techniques. Here, we describe an optimized method, including purifying away excessive primers from previous multiplex steps before singleplex real time detection, which dramatically increases the accuracy and robustness of the technique. Furthermore, we explain how performing the technique on a microfluidic chip at nanoliter volumes significantly reduces reagent costs and permits time effective high throughput miRNA expression profiling.

Here we describe an optimized multiplex reverse transcriptase quantitative PCR (qRT-PCR) protocol in combination with a microfluidic platform as a cost and time effective high-throughput screening tool for microRNA (miRNA) expression levels, especially when working with limited amounts of sample.

高通量微RNA分析:优化复纳升秤定量RT - PCR Fluidig​​m公司动态ArrayTM国际金融中心

The broad involvement of miRNAs in critical processes underlying development, tissue homoeostasis and disease has led to a surging interest among the ...

科研

生物工程

Single-cell Gene Expression Using Multiplex RT-qPCR to Characterize Heterogeneity of Rare Lymphoid Populations

Gene expression heterogeneity is an interesting feature to investigate in lymphoid populations. Gene expression in these cells varies during cell activation, stress, or stimulation. Single-cell multiplex gene expression enables the simultaneous assessment of tens of genes1,2,3. At the single-cell level, multiplex gene expression determines population heterogeneity4,5. It allows for the distinction of population heterogeneity by determining both the probable mix of diverse precursor stages among mature cells and also the diversity of cell responses to stimuli.
Innate lymphoid cells (ILC) have been recently described as a population of innate effectors of the immune response6,7. In this protocol, cell heterogeneity of the ILC hepatic compartment is investigated during homeostasis.
Currently, the most widely used technique to assess gene expression is RT-qPCR. This method measures gene expression only one gene at a time. Additionally, this method cannot estimate heterogeneity of gene expression, since multiple cells are needed for one test. This leads to the measurement of the average gene expression of the population. When assessing large numbers of genes, RT-qPCR becomes a time-, reagent-, and sample-consuming method. Hence, the trade-offs limit the number of genes or cell populations that can be evaluated, increasing the risk of missing the global picture.
This manuscript describes how single-cell multiplex RT-qPCR can be used to overcome these limitations. This technique has benefited from recent microfluidics technological advances1,2. Reactions occurring in multiplex RT-qPCR chips do not exceed the nanoliter-level. Hence, single-cell gene expression, as well as simultaneous multiple gene expression, can be performed in a reagent-, sample-, and cost-effective manner. It is possible to test cell gene signature heterogeneity at the clonal level between cell subsets within a population at different developmental stages or under different conditions4,5. Working on rare populations with large numbers of conditions at the single-cell level is no longer a restriction.

This protocol describes how to assess the expression of a large array of genes at the clonal level. Single-cell RT-qPCR produces highly reliable results with a strong sensitivity for hundreds of samples and genes.

单细胞基因表达利用多重RT-qPCR的表征罕见的淋巴人群的异质性

Gene expression heterogeneity is an interesting feature to investigate in lymphoid populations. Gene expression in these cells varies during cell ...

遗传学

Single-cell Gene Expression Profiling Using FACS and qPCR with Internal Standards

Gene expression measurements from bulk populations of cells can obscure the considerable transcriptomic variation of individual cells within those populations. Single-cell gene expression measurements can help assess the role of noise in gene expression, identify correlations in the expression of pairs of genes, and reveal subpopulations of cells that respond differently to a stimulus. Here, we describe a procedure to measure the expression of up to 96 genes in single mammalian cells isolated from a population growing in tissue culture. Cells are sorted into lysis buffer by fluorescence-activated cell sorting (FACS), and the mRNA species of interest are reverse-transcribed and amplified. Gene expression is then measured using a microfluidic real-time PCR machine, which performs up to 96 qPCR assays on up to 96 samples at a time. We also describe the generation and use of PCR amplicon standards to enable the estimation of the absolute number of each transcript. Compared with other methods of measuring gene expression in single cells, this approach allows for the quantification of more distinct transcripts than RNA FISH at a lower cost than RNA-Seq.

We describe a method to sort single mammalian cells and to quantify the expression of up to 96 target genes of interest in each cell. This method includes the use of internal qPCR standards to enable the estimation of absolute transcript counts.

单细胞基因表达谱使用流式细胞仪和qPCR内部标准

Gene expression measurements from bulk populations of cells can obscure the considerable transcriptomic variation of individual cells within those ...

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 ...

癌症研究

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 ...

Microarray-based Identification of Individual HERV Loci Expression: Application to Biomarker Discovery in Prostate Cancer

The prostate-specific antigen (PSA) is the main diagnostic biomarker for prostate cancer in clinical use, but it lacks specificity and sensitivity, particularly in low dosage values1&#8203;&#8203;. &#8216;How to use PSA&#39; remains a current issue, either for diagnosis as a gray zone corresponding to a concentration in serum of 2.5-10 ng/ml which does not allow a clear differentiation to be made between cancer and noncancer2 or for patient follow-up as analysis of post-operative PSA kinetic parameters can pose considerable challenges for their practical application3,4. Alternatively, noncoding RNAs (ncRNAs) are emerging as key molecules in human cancer, with the potential to serve as novel markers of disease, e.g. PCA3 in prostate cancer5,6 and to reveal uncharacterized aspects of tumor biology. Moreover, data from the ENCODE project published in 2012 showed that different RNA types cover about 62% of the genome. It also appears that the amount of transcriptional regulatory motifs is at least 4.5x higher than the one corresponding to protein-coding exons. Thus, long terminal repeats (LTRs) of human endogenous retroviruses (HERVs) constitute a wide range of putative/candidate transcriptional regulatory sequences, as it is their primary function in infectious retroviruses. HERVs, which are spread throughout the human genome, originate from ancestral and independent infections within the germ line, followed by copy-paste propagation processes and leading to multicopy families occupying 8% of the human genome (note that exons span 2% of our genome). Some HERV loci still express proteins that have been associated with several pathologies including cancer7-10. We have designed a high-density microarray, in Affymetrix format, aiming to optimally characterize individual HERV loci expression, in order to better understand whether they can be active, if they drive ncRNA transcription or modulate coding gene expression. This tool has been applied in the prostate cancer field (Figure 1).

Human endogenous retroviruses (HERV), which occupy 8% of the human genome, retain scarce coding capacities but a hundred thousand long terminal repeats (LTRs). A custom Affymetrix microarray was designed to identify individual HERV locus expression and was used on prostate cancer tissues as a proof of concept for future clinical studies.

应用生物标志物发现在前列腺癌:个别赫尔维位点表达微阵列鉴定

The prostate-specific antigen (PSA) is the main diagnostic biomarker for prostate cancer in clinical use, but it lacks specificity and sensitivity, ...

医学

Profiling of Estrogen-regulated MicroRNAs in Breast Cancer Cells

Estrogen plays vital roles in mammary gland development and breast cancer progression. It mediates its function by binding to and activating the estrogen receptors (ERs), ER&#945;, and ER&#946;. ER&#945; is frequently upregulated in breast cancer and drives the proliferation of breast cancer cells. The ERs function as transcription factors and regulate gene expression. Whereas ER&#945;&#39;s regulation of protein-coding genes is well established, its regulation of noncoding microRNA (miRNA) is less explored. miRNAs play a major role in the post-transcriptional regulation of genes, inhibiting their translation or degrading their mRNA. miRNAs can function as oncogenes or tumor suppressors&#160;and are also promising biomarkers. Among the miRNA assays available, microarray and quantitative real-time polymerase chain reaction (qPCR) have been extensively used to detect and quantify miRNA levels. To identify miRNAs regulated by estrogen signaling in breast cancer, their expression in ER&#945;-positive breast cancer cell lines were compared before and after estrogen-activation using both the &#181;Paraflo-microfluidic microarrays and Dual Labeled Probes-low density arrays. Results were validated using specific qPCR assays, applying both Cyanine dye-based and Dual Labeled Probes-based chemistry. Furthermore, a time-point assay was used to identify regulations over time. Advantages of the miRNA assay approach used in this study is that it enables a fast screening of mature miRNA regulations in numerous samples, even with limited sample amounts. The layout, including the specific conditions for cell culture and estrogen treatment, biological and technical replicates, and large-scale screening followed by in-depth confirmations using separate techniques, ensures a robust detection of miRNA regulations,&#160;and eliminates false positives and other artifacts. However, mutated or unknown miRNAs, or regulations at the primary and precursor transcript level, will not be detected. The method presented here represents a thorough investigation of estrogen-mediated miRNA regulation.

Molecular signaling through both estrogen and microRNAs are critical in breast cancer development and growth. Estrogen activates the estrogen receptors, which are transcription factors. Many transcription factors can regulate the expression of microRNAs, and estrogen-regulated microRNAs can be profiled using different large-scale techniques.

在乳腺癌细胞雌激素调节小分子RNA的剖析

Estrogen plays vital roles in mammary gland development and breast cancer progression. It mediates its function by binding to and activating the estrogen ...

miRNA Expression Analyses in Prostate Cancer Clinical Tissues

A critical challenge in prostate cancer (PCa) clinical management is posed by the inadequacy of currently used biomarkers for disease screening, diagnosis, prognosis and treatment. In recent years, microRNAs (miRNAs) have emerged as promising alternate biomarkers for prostate cancer diagnosis and prognosis. However, the development of miRNAs as effective biomarkers for prostate cancer heavily relies on their accurate detection in clinical tissues. miRNA analyses in prostate cancer clinical specimens is often challenging owing to tumor heterogeneity, sampling errors, stromal contamination etc. The goal of this article is to describe a simplified workflow for miRNA analyses in archived FFPE or fresh frozen prostate cancer clinical specimens using a combination of quantitative real-time PCR (RT-PCR) and in situ hybridization (ISH). Within this workflow, we optimize the existing methodologies for miRNA extraction from FFPE and frozen prostate tissues and expression analyses by Taqman-probe based miRNA RT-PCR. In addition, we describe an optimized method for ISH analyses formiRNA detection in prostate tissues using locked nucleic acid (LNA)- based probes. Our optimized miRNA ISH protocol can be applied to prostate cancer tissue slides or prostate cancer tissue microarrays (TMA).

Here we describe a simplified protocol for microRNA (miRNA) expression analyses in archived Formalin-Fixed, Paraffin-Embedded (FFPE) or fresh frozen prostate cancer (PCa) clinical tissues employing quantitative real-time PCR (RT-PCR) and in situ hybridization (ISH).

在前列腺癌的临床组织中miRNA的表达分析

A critical challenge in prostate cancer (PCa) clinical management is posed by the inadequacy of currently used biomarkers for disease screening, ...

Evaluation of Lung Metastasis in Mouse Mammary Tumor Models by Quantitative Real-time PCR

Metastatic disease is the spread of malignant tumor cells from the primary cancer site to a distant organ and is the primary cause of cancer associated death 1. Common sites of metastatic spread include lung, lymph node, brain, and bone 2. Mechanisms that drive metastasis are intense areas of cancer research. Consequently, effective assays to measure metastatic burden in distant sites of metastasis are instrumental for cancer research. Evaluation of lung metastases in mammary tumor models is generally performed by gross qualitative observation of lung tissue following dissection. Quantitative methods of evaluating metastasis are currently limited to ex vivo and in vivo imaging based techniques that require user defined parameters. Many of these techniques are at the whole organism level rather than the cellular level 3-6. Although newer imaging methods utilizing multi-photon microscopy are able to evaluate metastasis at the cellular level 7, these highly elegant procedures are more suited to evaluating mechanisms of dissemination rather than quantitative assessment of metastatic burden. Here, a simple in vitro method to quantitatively assess metastasis is presented. Using quantitative Real-time PCR (QRT-PCR), tumor cell specific mRNA can be detected within the mouse lung tissue.

This protocol describes how to use quantitative Real-time PCR (QRT-PCR) to detect tumor cell specific mRNA representing metastasis within the mouse lung tissue.

评价肺转移的小鼠乳腺肿瘤模型定量实时荧光定量PCR

Metastatic disease is the spread of malignant tumor cells from the primary cancer site to a distant organ and is the primary cause of cancer associated ...

Clinicopathological Analysis of miRNA Expression in Breast Cancer Tissues by Using miRNA In Situ Hybridization

In this article, we describe a detailed protocol for miRNA detection in breast cancer tissue using in situ hybridization with a digoxigenin-labelled LNA (Locked Nucleic Acid) probe. The probe was recognized by anti-DIG alkaline phosphatase antibodies and later developed using alkaline phosphatase substrate producing fluorescence signals. Here we utilized miRNA in situ hybridization (MISH) technique to analyze expression of miR-489 in tissues from breast cancer patients. This technique can detect the localization of miRNA of interest in individual tissue samples. This technique can be used to compare the expression of desired miRNA in tumor tissue with that in adjacent normal tissue and to identify the specific structures responsible for expressing this miRNA. This technique can be very useful in answering certain clinical questions, such as role of specific miRNA in the development of cancer. Our results indicate that mammary epithelial cells express significantly higher levels of miR-489 than adjacent tumor cells.

Clinicopathological Analysis of miRNA Expression in Breast Cancer Tissues by Using miRNA In Situ Hybridization

Here we present a protocol to detect miRNA expression in breast cancer patient samples using miRNA in situ hybridization.

利用miRNA的miRNA表达的临床病理分析乳腺癌组织<em&gt;原位</em&gt;杂交

In this article, we describe a detailed protocol for miRNA detection in breast cancer tissue using in situ hybridization with a digoxigenin-labelled LNA ...

应用乳腺癌档案材料的多路 RNA 表达方法的优化

摘要

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高通量微RNA分析:优化复纳升秤定量RT - PCR Fluidigm公司动态ArrayTM国际金融中心

单细胞基因表达利用多重RT-qPCR的表征罕见的淋巴人群的异质性

单细胞基因表达谱使用流式细胞仪和qPCR内部标准

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

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

应用生物标志物发现在前列腺癌:个别赫尔维位点表达微阵列鉴定

在乳腺癌细胞雌激素调节小分子RNA的剖析

在前列腺癌的临床组织中miRNA的表达分析

评价肺转移的小鼠乳腺肿瘤模型定量实时荧光定量PCR

利用miRNA的miRNA表达的临床病理分析乳腺癌组织

应用乳腺癌档案材料的多路 RNA 表达方法的优化

摘要

探索更多视频

此视频中的章节

高通量微RNA分析:优化复纳升秤定量RT - PCR Fluidig​​m公司动态ArrayTM国际金融中心

单细胞基因表达利用多重RT-qPCR的表征罕见的淋巴人群的异质性

单细胞基因表达谱使用流式细胞仪和qPCR内部标准

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

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

应用生物标志物发现在前列腺癌:个别赫尔维位点表达微阵列鉴定

在乳腺癌细胞雌激素调节小分子RNA的剖析

在前列腺癌的临床组织中miRNA的表达分析

评价肺转移的小鼠乳腺肿瘤模型定量实时荧光定量PCR

利用miRNA的miRNA表达的临床病理分析乳腺癌组织

高通量微RNA分析:优化复纳升秤定量RT - PCR Fluidigm公司动态ArrayTM国际金融中心