本研究中使用的人体肿瘤样本是根据人类研究的当地道德准则, deidentified 和从商业来源获得的。
1. 样品、设备和试剂的制备
<ol><li>
FFPE 样品制备
	<ol>
<li>组织<ol>
<li>紧接着解剖后, 固定的组织 (切割成块的3–4毫米厚度) 在10% 中性缓冲福尔马林 (NBF) 的16–32个 h 在室温下 (RT)。 注: 固定时间根据组织类型和大小而定。</li>
			<li>使用标准乙醇 (乙醇) 系列 (70% 乙醇为30–60最小, 80% 乙醇30–60最小, 90% 乙醇30–60最小, 95% 乙醇为30–60最小, 3x 100% 乙醇30–60分钟) 和二甲苯, 用1x 磷酸盐缓冲盐水冲洗样品。</li>
			<li>使用标准程序6将样品嵌入石蜡切片, 并根据需要修剪石蜡块以除去多余的石蜡。 注意: 块大小会因组织样本大小而异, 但典型大小为 0.75 x 0.75 英寸2或更小。</li>
		</ol>
</li>
		<li>单元格线<ol>
<li>根据特定细胞线的推荐方法收集和颗粒细胞。</li>
			<li>固定细胞在10% 甲醛在 RT 24 小时的转子。</li>
			<li>在 prewarmed 处理凝胶 (Histogel) 中制备细胞作为颗粒。通过将凝胶细胞颗粒放在冰上的 parafilm 上, 使颗粒凝固, 并让它坐2–3分钟. 在 1x PBS 中浸泡凝胶细胞颗粒。</li>
			<li>脱水和嵌入细胞丸, 如步骤1.1.1 所述。</li>
		</ol>
</li>
		<li>部分准备工作<ol>
<li>使用切片将嵌入的组织/细胞切成5到1微米的部分, 安装在静电胶玻璃滑梯上的部分, 并在一夜间在 RT 上风干。 注: 可在干燥处贮存3月以上的幻灯片。</li>
			<li>将幻灯片放在幻灯片机架中, 然后在进行化验之前, 在60摄氏度的循环空气烘箱中烘烤已安装的组织幻灯片, 1 小时。 注: 立即使用幻灯片或将它们存储在 RT 上, 干燥剂长达1周。长时间贮存可能导致 RNA 降解。</li>
		</ol>
</li>
	</ol>
</li>
	<li>
设备准备
	<ol>
<li>将杂交烤箱设置为40摄氏度。彻底润湿加湿纸, 除去任何残留 dH2o 将纸张插入湿度控制托盘中, 并在使用前将纸盒插入到杂交烤箱中, prewarm 至少30分钟。</li>
	</ol>
</li>
	<li>
试剂制剂
	<ol>
<li>用200毫升二甲苯填充两个清洗剂菜肴, 并用200毫升100% 乙醇填充两个染色盘, 将用于 deparaffinization 的切片。</li>
		<li>200毫升的商用1x 目标检索试剂, 添加180毫升的 dH2O 到20毫升10x 目标检索试剂。将两个幻灯片持有人放在蒸笼中。用200毫升的1x 目标检索试剂填充一张幻灯片持有者, 并用200毫升的 dH2o 填充其他滑轨, 加热两种溶液, 用蒸笼煮沸。</li>
		<li>温暖的50x 洗涤缓冲器到40°c 为 10–20 min. 通过稀释60毫升的 prewarmed 50x 洗涤缓冲器与 dH2O 的2.94 升来准备3升1x 洗涤缓冲器。</li>
		<li>在油烟机中, 准备50% 鳃的苏木精 counterstaining 溶液, 加入100毫升鳃的苏木精到100毫升的 dH2O。在油烟机中, 通过加入1.43 毫升的28–30% 氢氧化铵到250毫升的 dH2O, 制备发蓝试剂 (0.02% (w/v) 氨水)。</li>
		<li>Prewarm 目标探针在40°c 10 分钟之前探针杂交并且带来放大试剂 (安培 0–6) 到 RT。</li>
	</ol>
</li>
</ol>2. RNA原位杂交法
<ol><li>
Deparaffinization 脱水
	<ol>
<li>烘烤后, 如步骤 1.1.3.2 (可选停止点 1) 中所述, deparaffinize 的部分二甲苯5分钟与搅拌。Deparaffinize 再次在新鲜二甲苯5分钟脱水100% 乙醇2分钟与搅拌, 并再次重复在新鲜100% 乙醇2分钟。</li>
		<li>空气干燥的幻灯片5分钟在60°c 在循环空气烤箱或在 RT, 直到他们完全干燥 (可选停止点 2)。</li>
	</ol>
</li>
	<li>
样品预处理
	<ol>
<li>在 RT 中孵化出4滴可供使用的过氧化氢, 用于10分钟, 以淬火内源过氧化物酶活性。醒酒的解决方案从幻灯片和冲洗两次与 dH2O。</li>
		<li>用200毫升的目标检索试剂在一艘蒸笼的100摄氏度孵育15–30。 注: 潜伏期可能会因组织类型而异。在该协议中, 对肿瘤标本和细胞颗粒进行了15分钟的目标检索。</li>
		<li>醒酒的解决方案从幻灯片, 冲洗两次与 dH2O, 蘸100% 乙醇3分钟, 并干燥的幻灯片在60°c 在循环空气烤箱或 RT, 直到完全干燥。</li>
		<li>使用疏水性钢笔在截面周围画一个疏水屏障, 大约 0.75 x 0.75 英寸2。 注意: 建议不要绘制较小的屏障。对于较大的部分, 需要绘制更大的屏障。</li>
		<li>让屏障完全干燥1分钟, 或在 RT 过夜 (可选停止点 3)。</li>
		<li>将幻灯片放在幻灯片夹中, 并将幻灯片夹放在湿度控制托盘中。加入4滴蛋白酶 III 到每张幻灯片和孵化他们为15–30分钟在40°c 在杂交烤箱蛋白质消化。 注: 潜伏期可能会因组织类型而异。在本协议中, 对肿瘤标本进行蛋白酶消化30分钟, 细胞微丸15分钟。</li>
		<li>醒酒的解决方案从幻灯片和冲洗两次与 dH2O。</li>
	</ol>
</li>
	<li>
探针杂交
	<ol>
<li>添加〜4滴适当的现成的探头解决方案覆盖整个部分。如果使用较大的部分, 添加〜5–6滴。</li>
		<li>杂交探针为2小时在40°c 在杂交烤箱。醒酒的解决方案从幻灯片和洗涤200毫升1x 洗涤缓冲的幻灯片, 2 分钟的 RT 与偶尔的鼓动。在此步骤中重复洗涤过程。</li>
	</ol>
</li>
	<li>
信号放大
	<ol>
<li>孵化的部分与〜4滴安培0每张幻灯片在40°c 的杂交烤箱30分钟醒酒解决方案, 并清洗200毫升1x 洗涤缓冲器的幻灯片, 在 RT 与偶尔的搅拌。在此步骤中重复洗涤过程。</li>
		<li>孵化的部分与〜4滴安培1每张幻灯片在40°c 的杂交烤箱15分钟醒酒解决方案, 并清洗200毫升1x 洗涤缓冲器的幻灯片, 在 RT 与偶尔的搅拌。重复洗涤过程。</li>
		<li>孵化的部分与〜4滴安培2每张幻灯片在40°c 的杂交烤箱30分钟醒酒解决方案, 并清洗200毫升1x 洗涤缓冲器的幻灯片, 在 RT 与偶尔的搅拌。重复洗涤过程。</li>
		<li>孵化的部分与〜4滴安培3每张幻灯片在40°c 的杂交烤箱30分钟醒酒解决方案, 并清洗200毫升1x 洗涤缓冲器的幻灯片, 在 RT 与偶尔的搅拌。重复洗涤过程。</li>
		<li>孵化的部分与〜4滴安培4每张幻灯片在40°c 的杂交烤箱15分钟醒酒解决方案, 并清洗200毫升1x 洗涤缓冲器的幻灯片, 在 RT 与偶尔的搅拌。重复洗涤过程。</li>
		<li>每张幻灯片上的4滴安培为30分钟, 在 rt 中孵化出5醒酒的切片, 并以偶尔搅拌的方式在 rt 上清洗200毫升1x 洗涤缓冲器中的幻灯片。重复洗涤过程。</li>
		<li>每张幻灯片上的4滴安培为15分钟, 在 rt 中孵化出6醒酒的切片, 并以偶尔搅拌的方式在 rt 上清洗200毫升1x 洗涤缓冲器中的幻灯片。重复洗涤过程。</li>
	</ol>
</li>
	<li>
信号检测
	<ol>
<li>准备快速的红色染料工作溶液。对于一个 0.75 x 0.75 英寸2屏障的幻灯片, 添加 2 ul 的快速红 B 染料到 120 ul 的快速红 a 入管和混合良好。 注: 根据疏水屏障的大小和幻灯片的数量, 快速红色工作溶液的体积会有所不同。</li>
		<li>醒酒从幻灯片中的多余液体, 并添加快速红色染料工作解决方案的幻灯片。用盖子在托盘上孵化10分钟的幻灯片, 以避免光线暴露。 注: 使用快速红色工作溶液在5分钟内准备, 并不要暴露它直接阳光或紫外线。</li>
		<li>醒酒快速红色溶液, 用自来水冲洗两次幻灯片。将幻灯片放回幻灯片架中。</li>
	</ol>
</li>
	<li>
Counterstaining
	<ol>
<li>Counterstain 组织切片, 50% 鳃的苏木精溶液为2分钟的 RT. 用自来水冲洗幻灯片, 重复多次, 直到幻灯片清晰, 而各部分保持紫色。将滑块浸入0.02% 氨水中发蓝 (浸2–3倍)。用自来水代替氨水, 3–5时间冲洗滑梯。</li>
	</ol>
</li>
	<li>
幻灯片安装
	<ol>
<li>将循环空气烘箱中的幻灯片干燥60摄氏度, 15 分钟或 RT, 直至完全干燥。在每张幻灯片上放置一滴固定试剂, 并将盖玻片放在每个切片上。避免气泡的任何陷阱。空气干燥幻灯片至少5分钟。 注: 快速红色衬底是酒精敏感。不要在酒精中脱水。</li>
	</ol>
</li>
	<li>
可视 化
	<ol>
<li>在标准 brightfield 显微镜下观察幻灯片。</li>
	</ol>
</li>
</ol>

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所有作者均采用高级细胞诊断公司。

在本报告中, 详细讨论了新的检测协议及其应用。该方法可以直接可视化外显子结, 短靶和高度同源序列, 和点突变的组织背景。该检测基于 RNAscope 技术5 , 因此能够进行单分子检测。然而, 由于一个先进的放大系统, 信号可以检测到, 探头包含多达一个双 Z 对或与目标模板长度只有50核苷酸。由于探头的长度可能短于单个双 Z, 因此可以检测外显子连接、短目标和高度同源序列以及点突变 (表 1)。
为了成功地进行化验, 有几个技术建议。首先, 组织应固定在新鲜的10% 中性缓冲福尔马林 (NBF) 室温16–32个 h10。Underfixation (< 16 小时) 或 overfixation (> 32 h) 将损害化验的性能, 可能需要额外的优化。第二, 为了确保对鲁棒探针杂交和信号放大所需的温度和湿度进行最佳控制, 应将滑动处理系统和杂交烤箱用于协议步骤2.3 至 5 (蛋白酶预处理、探针杂交、信号放大和信号检测)。第三, 多余的剩余缓冲器应该在整个协议的每一步之前正确排水 (但不要太多, 以使组织切片干燥)。如果幻灯片干燥, 重要的非特定信号将会发展。第四, 根据组织类型, 预处理优化可能是必要的。使用错误的蛋白酶或表现为一个不理想的时间长度可能导致在或过度消化, 并会对信号产生负面影响。最后, 必须始终使用测试探头运行正向和负控制。负控制探头确保没有背景信号, 正向控制探头确保检测结果正确无误, 样品中的 RNA 质量是最佳的解释测试探针效果的方法。如果在正控探针上没有信号, 则样本中的 RNA 质量可能是次优, 而测试探针可能无法看到信号。
除了手工化验, 还证明了在自动 stainers 上进行化验的能力 (图 6)。这种自动化的检测产生高信噪比, 适用于相同的应用如表 1所示;然而, 自动化化验的好处包括化验条件的标准化、用户间变异性的最小化和实际使用时间, 以及以可靠的方式对组织样本进行高通量筛选的津贴。
虽然免疫组化 (IHC) 和 qRT PCR 检测的剪接变种 (特别是 EGFRvIII), 在 FFPE 临床标本, 这些技术可能缺乏必要的特异性, 并没有提供洞察的空间分辨率的拼接变体表达式, 分别为11、12。该方法的一个关键优点是它对剪接结的高度敏感和特异的可视化, 同时保持形态学组织的上下文。在这里, 该化验的能力, 精确目标的独特的外显子连接的几个剪接变体, 包括 EGFRvIII 和 METΔ14, 被证明 (图3和 6)。此外, 该检测结果显示, 检测前列腺癌的剪接变异 AR-V7, 脑中 ErbB4 的多重亚型, 并确认在小鼠脑3,13,14的圆形 RNA Cdr1as 的挖空。
通过这种方法进行短目标序列检测, 可以直观地显示 RNA 序列的长度为50核苷酸, 这是从 Jurkat 细胞中提取的 CDR3 序列的检测结果 (图 3)。该检测还可以检测与其他家庭成员或物种高度同源的基因序列, 如 Revêchon 所示, 后者使用短靶检测方法检测在小鼠皮下白脂肪组织中表达的人 progerin15。此外, 小核仁 RNA (snoRNA), CRISPR 介导的基因编辑, 和前体-microRNA 可以检测原位与短靶检测。最近, 傅等人将这一方法与 IHC 相结合, 以确定视网膜上的精确细胞, 表达前 miRNA mir125b16。
肿瘤的突变分析是研究肿瘤进展和发展靶向治疗的关键。虽然通过高通量测序可以实现突变分析, 但这种技术不能完全解决肿瘤的异质性, 也不能将基因改变与细胞形态学17,18联系起来。使用这种方法检测点突变可以区分 RNA 目标序列在单基分辨率, 这是通过检测 EGFR L858R 和 KRAS G12A 单核苷酸变化的细胞系 (图 5) 验证的。此外, 贝克等人用点突变法对大肠癌 BRAF、KRAS 和 PIK3CA 癌基因中的多项突变进行了靶向分析18。他们能够识别和空间地映射罕见的肿瘤细胞突变体 subclones, 最终显示它们是如何促进肿瘤内异质性的。
总之, 研制了一种专门的 RNA 检测方法。这种方法允许检测拼接变形, 短序列和原位突变。它是敏感的, 具体的, 可量化的, 并适用于性能的手动方法和自动 stainers。

高通量 transcriptomic 技术, 如微阵列和下一代 RNA 测序 (RNAseq) 已成倍地提高了发现 RNA 生物标志物的诊断, 预后和预测临床价值的各种疾病, 包括癌症1,2。为了促进这些生物标志物在精密医学中的应用, 需要标准化和健壮的技术来测量临床样品的组织环境中的 RNA 生物标志物。尽管广泛建立了像 RNAseq 和定量 rt-pcr 这样的研磨和结合检测技术, 使高度敏感的基因表达测量, 所需的组织均匀化和 RNA 分离意味着体内细胞型特异性的丧失和形态学信息3。传统的原位RNA 检测方法缺乏可靠测量组织环境4中稀有或低表达 rna 生物标志物所需的灵敏度和特异性。
一种商业化的原位杂交 (RNAscope) 检测技术是解决这些挑战的一项新技术, 同时也使单个 RNA 分子的高度敏感和特异的可视化程度大于300核苷酸在组织形态学上下文中。这种类型的检测使用一个独特的寡核苷酸探针设计约6–20双 Z 探针对结合先进的杂交为基础的信号放大5。
本研究描述了一种专门的 rna 测试, BaseScope, 补充了以前设计的商业技术, 可以检测 RNA 目标序列短于50核苷酸在单核苷酸分辨率。这种分析解决了复杂的转录, 适用于准确检测外显子结, 短靶序列, 和点突变的组织上下文 (表 1) 使用尽可能少的一个双 Z 探针对。本报告说明了完整的检测协议及其在检测剪接变种、T 细胞受体克隆的 CDR3 序列以及 FFPE 细胞系和肿瘤组织中的单核苷酸突变方面的应用。

<table><tbody><tr><td>HybEZ Oven (110 or 220 VAC) or HybEZ II Oven (110 or 220V)</td><td>ACD</td><td>310010 or 310013 (HybEZ&amp;trade;), 321710 or 321720 (HybEZ&amp;trade; II)</td><td></td></tr><tr><td>HybEZ Humidity Control Tray (with lid)</td><td>ACD</td><td>310012</td><td></td></tr><tr><td>ACD EZ-Batch Slide Rack (20 slide capacity) 1 rack 310017</td><td>ACD</td><td>310017</td><td></td></tr><tr><td>HybEZ Humidifying Paper</td><td>ACD</td><td>310015</td><td></td></tr><tr><td>ImmEdge Hydrophobic Barrier Pen (required)&amp;nbsp;</td><td>Vector Laboratory&amp;nbsp;</td><td>H-4000</td><td></td></tr><tr><td>SuperFrost Plus Slides (required)&amp;nbsp;</td><td>Fisher Scientific&amp;nbsp;</td><td>12-550-15</td><td></td></tr><tr><td>10% neutral-buffered formalin (NBF)&amp;nbsp;</td><td>MLS</td><td>/</td><td>MLS Major Laboratory Supplier in North America. For other regions, please check Catalog Numbers with your local lab supplier.</td></tr><tr><td>Paraffin wax</td><td>MLS</td><td>/</td><td>MLS Major Laboratory Supplier in North America. For other regions, please check Catalog Numbers with your local lab supplier.</td></tr><tr><td>Microtome</td><td>MLS</td><td>/</td><td>MLS Major Laboratory Supplier in North America. For other regions, please check Catalog Numbers with your local lab supplier.</td></tr><tr><td>Gill&amp;rsquo;s Hematoxylin I&amp;nbsp;</td><td>American Master Tech Scientific/MLS</td><td>&amp;nbsp;HXGHE1LT</td><td>MLS Major Laboratory Supplier in North America. For other regions, please check Catalog Numbers with your local lab supplier.</td></tr><tr><td>Xylene&amp;nbsp;</td><td>Fisher Scientific/MLS</td><td>X3P-1GAL</td><td>MLS Major Laboratory Supplier in North America. For other regions, please check Catalog Numbers with your local lab supplier.</td></tr><tr><td>Tissue-Tek Vertical 24 Slide Rack&amp;nbsp;</td><td>American Master Tech Scientific/MLS</td><td>LWSRA24</td><td>MLS Major Laboratory Supplier in North America. For other regions, please check Catalog Numbers with your local lab supplier.</td></tr><tr><td>Tissue-Tek Staining Dishes&amp;nbsp;</td><td>American Master Tech Scientific/MLS</td><td>LWT4457EA</td><td>MLS Major Laboratory Supplier in North America. For other regions, please check Catalog Numbers with your local lab supplier.</td></tr><tr><td>Tissue-Tek Clearing Agent Dishes, xylene resistant&amp;nbsp;</td><td>American Master Tech Scientific/MLS</td><td>LWT4456EA</td><td>MLS Major Laboratory Supplier in North America. For other regions, please check Catalog Numbers with your local lab supplier.</td></tr><tr><td>100% alcohol (EtOH)&amp;nbsp;</td><td>American Master Tech Scientific/MLS</td><td>ALREACS</td><td>MLS Major Laboratory Supplier in North America. For other regions, please check Catalog Numbers with your local lab supplier.</td></tr><tr><td>VectaMount Permanent Mounting Medium (required)</td><td>Vector Labs&amp;nbsp;</td><td>H-5000</td><td></td></tr><tr><td>Cover Glass, 24 mm x 50 mm</td><td>Fisher Scientific/MLS&amp;nbsp;</td><td>12-545-F</td><td>MLS Major Laboratory Supplier in North America. For other regions, please check Catalog Numbers with your local lab supplier.</td></tr><tr><td>Ammonium hydroxide, 28&amp;ndash;30%</td><td>Sigma-Aldrich/MLS</td><td>320145-500mL</td><td>MLS Major Laboratory Supplier in North America. For other regions, please check Catalog Numbers with your local lab supplier.</td></tr><tr><td>Carboy (&gt;3L)</td><td>MLS</td><td>/</td><td>MLS Major Laboratory Supplier in North America. For other regions, please check Catalog Numbers with your local lab supplier.</td></tr><tr><td>Oster Steamer Model 5712, Black and Decker Steamer HS3000, or the Braun Multiquick FS 20 Steamer</td><td>/</td><td>/</td><td></td></tr><tr><td>Digital thermometer</td><td>MLS</td><td>/</td><td>MLS Major Laboratory Supplier in North America. For other regions, please check Catalog Numbers with your local lab supplier.</td></tr><tr><td>Water bath or incubator, capable of holding temperature at 40 +/&amp;ndash; 1 &amp;deg;C</td><td>MLS</td><td>/</td><td>MLS Major Laboratory Supplier in North America. For other regions, please check Catalog Numbers with your local lab supplier.</td></tr><tr><td>Pipettors and tips, 1&amp;ndash;1,000 &amp;mu;L</td><td>MLS</td><td>/</td><td>MLS Major Laboratory Supplier in North America. For other regions, please check Catalog Numbers with your local lab supplier.</td></tr><tr><td>Distilled water</td><td>MLS</td><td>/</td><td>MLS Major Laboratory Supplier in North America. For other regions, please check Catalog Numbers with your local lab supplier.</td></tr><tr><td>Tubes (various sizes)</td><td>MLS</td><td>/</td><td>MLS Major Laboratory Supplier in North America. For other regions, please check Catalog Numbers with your local lab supplier.</td></tr><tr><td>Fume hood</td><td>MLS</td><td>/</td><td>MLS Major Laboratory Supplier in North America. For other regions, please check Catalog Numbers with your local lab supplier.</td></tr><tr><td>Graduated cylinder</td><td>MLS</td><td>/</td><td>MLS Major Laboratory Supplier in North America. For other regions, please check Catalog Numbers with your local lab supplier.</td></tr><tr><td>Parafilm</td><td>MLS</td><td>/</td><td>MLS Major Laboratory Supplier in North America. For other regions, please check Catalog Numbers with your local lab supplier.</td></tr><tr><td>Paper towel or absorbent paper</td><td>MLS</td><td>/</td><td>MLS Major Laboratory Supplier in North America. For other regions, please check Catalog Numbers with your local lab supplier.</td></tr><tr><td>Microcentrifuge</td><td>MLS</td><td>/</td><td>MLS Major Laboratory Supplier in North America. For other regions, please check Catalog Numbers with your local lab supplier.</td></tr><tr><td>Microscope and accessories</td><td>MLS</td><td>/</td><td>MLS Major Laboratory Supplier in North America. For other regions, please check Catalog Numbers with your local lab supplier.</td></tr><tr><td>Drying oven, capable of holding temperature at 60 +/&amp;ndash; 1 &amp;deg;C</td><td>MLS</td><td>/</td><td>MLS Major Laboratory Supplier in North America. For other regions, please check Catalog Numbers with your local lab supplier.</td></tr><tr><td>Formaldehyde</td><td>MLS</td><td>/</td><td>MLS Major Laboratory Supplier in North America. For other regions, please check Catalog Numbers with your local lab supplier.</td></tr><tr><td>Histogel</td><td>Fisher Scientific/MLS</td><td>22-110-678</td><td></td></tr><tr><td>BaseScope Reagent Kit - RED</td><td>Advanced Cell Diagnostics</td><td>322900</td><td></td></tr><tr><td>BaseScope Hs-EGFR-E1E2</td><td>Advanced Cell Diagnostics</td><td>701701</td><td></td></tr><tr><td>BaseScope Hs-EGFR-E1E8</td><td>Advanced Cell Diagnostics</td><td>701711</td><td></td></tr><tr><td>BaseScope Hs-EGFR-E7E8</td><td>Advanced Cell Diagnostics</td><td>701721</td><td></td></tr><tr><td>BaseScope Hs-EGFR-E8E9</td><td>Advanced Cell Diagnostics</td><td>701731</td><td></td></tr><tr><td>BaseScope Hs-MET-E14E15</td><td>Advanced Cell Diagnostics</td><td>701811</td><td></td></tr><tr><td>BaseScope Hs-MET-E13E15</td><td>Advanced Cell Diagnostics</td><td>701801</td><td></td></tr><tr><td>BaseScope Hs-KRAS-G12A</td><td>Advanced Cell Diagnostics</td><td>705491</td><td></td></tr><tr><td>BaseScope Hs-KRAS-G12-nt35WT</td><td>Advanced Cell Diagnostics</td><td>705531</td><td></td></tr><tr><td>BaseScope Hs-EGFR-L858R</td><td>Advanced Cell Diagnostics</td><td>705451</td><td></td></tr><tr><td>BaseScope Hs-EGFR-L858WT</td><td>Advanced Cell Diagnostics</td><td>705461</td><td></td></tr><tr><td>BaseScope Control Probe Pack Human</td><td>Advanced Cell Diagnostics</td><td>322975</td><td></td></tr></tbody></table>

visualizing genetic variants, short targets, and point mutations in the morphological tissue context with an rna in situ hybridization assay

由于精密医学高度依赖于对生物标志物的精确检测, 越来越需要标准化和健壮的技术来检测临床标本中的 RNA 生物标志物。虽然像 RNAseq 和定量 rt-pcr 这样的研磨和结合检测能够使高度敏感的基因表达测量, 但它们也需要 RNA 提取, 从而防止在形态学组织上下文中有价值的表达分析。这里描述的原位杂交方法可以在单核苷酸分辨率和单细胞水平上检测出 RNA 靶序列短于50核苷酸。这项化验是补充以前开发的商业化验, 并使敏感和特定的原位检测的剪接变种, 短靶点, 并在组织内的突变。在本协议中, 探针设计为针对两个临床重要的剪接变体, EGFRvIII 和 METΔ14的独特的外显子结。通过对 Jurkat t 细胞系 t 细胞受体α和β CDR3 序列的特异检测, 证明了短靶序列的检测。还显示了这一方法的效用, 以区别 RNA 目标序列在单核苷酸分辨率 (点突变) 通过可视化 EGFR L858R 和 KRAS G12A 单核苷酸变化的细胞系使用自动染色平台。总之, 该协议显示了一个专门的 RNA 的测试, 使检测的拼接变形, 短序列和原位突变的手工性能和自动 stainers。

原位杂交检测工作流: 该工作流描述在图 1中, 包括四部分: 通透的细胞或组织的目标检索和蛋白酶的解决方案, 对目标 RNA 的探针杂交, 信号放大, 信号的可视化。信号也可以使用数字成像软件系统或以半定量的方式量化, 基于每个单元格的点数。图 2中描述的手动程序在商用自动染色系统中也已完全自动化。
外显子结检测的代表性染色 (EGFRvIII 剪接变体): EGFRvIII 是表皮生长因子受体的变体, 它来源于外显子2到7的帧内基因组删除, 导致组成性主动致癌信号7.该方法用于鉴定 FFPE 胶质瘤 (肾小球) 肿瘤标本中的 EGFRvIII 状态。单双 Z 探头设计用于跨越外显子连接, 以检测小波、突变体或两个记录 (图 3A)。E1/E2 的 EGFR 探头横跨外显子1和 2 () 或外显子7和 8 (E7/E8) 的连接点, 而 EGFRvIII 特定探针横跨外显子1和 8 (E1/E8) 的交界处。一个横跨外显子8和 9 (E8/E9) 交界处的共同探针也被用来检测总的 EGFR (EGFRvIII 记录)。然后用所有探针测定 FFPE 肾小球标本中 EGFR 的状态。图 3B所示的两个代表性例子取自一项更大的研究。EGFR 的状态由一个独立的方法, rt-pcr 证实。两个小波探测器都检测到两个样本中的信号, 表明两个样本表示的是 EGFR (图 3B)。然而, 突变探针只显示 EGFRvIII + 样本中的信号检测, 确认这个样本确实是 EGFRvIII 变体的阳性 (图 3B, 左面板)。相反, 突变探针没有检测到 EGFRvIII 样本中的信号 (图 3B, 右面板)。结果表明, 外显子结检测可以识别肾小球 FFPE 肿瘤标本中的 EGFRvIII 状态。
短目标的代表性染色: CDR3, 或互补确定区域 3, 是一个高度可变的领域, T 细胞受体。通常, CDR3 序列相当短;例如, Jurkat T 细胞线的 CDR3 α和β序列分别为51和48核苷酸 (图 4A)。为了识别 Jurkat 细胞中表达的特定 CDR 序列, CDR3 α和β在 Jurkat T 细胞线中表达的反义探针被产生, 除了对 CDR3 α和β的感觉探针作为负控制探针。所有探针然后测试在 FFPE 准备好的 Jurkat 细胞与试验。在 Jurkat 细胞中, CDR3 α和β的抗感探针观察到了鲁棒染色, 而感觉探针检测到没有信号 (图 4B)。这些结果表明, 短期靶向检测的能力, 以辨别高度可变, 但短的 CDR 序列的 T 细胞受体克隆。
点突变的代表性染色 EGFR L858R: 点突变探针被开发来检测单核苷酸变异和小插入或删除 (INDELs) 在肿瘤背景下。图 5A展示了原位检测 L858R (2573T > G) 点突变的能力。设计了两个探头: 一种检测 L858R 突变的 egfr 序列, 另一种用于检测 egfr L858 序列。两种探针均经 FFPE 制备的细胞系测试: H2229, 仅表达 EGFR L858;和 H1975, 这是异型的 EGFR L858R 突变。L858R 突变探针检测到的信号仅在 H1975 细胞线, 而不是在 H2229 细胞线。然而, 探测器检测到两个细胞线上的信号。同样,图 5B可视化的点突变 KRAS G12A 的原位检测 (35 克 > C)。设计了两个探头来检测 KRAS G12A MT 和 KRAS G12 的小波序列, 然后在 HuT78 细胞线 (只表示 KRAS G12) 和 SW116 细胞线 (异型 KRAS 突变) 上进行测试。当 KRAS G12 探头检测到两个细胞线上的信号时, KRAS G12A 探头只检测到 SW116 细胞线上的信号。结合这些数据, 证明了点突变法检测细胞和组织环境中单核苷酸多态性的技术能力。
自动原位检测的代表性染色: 自动化测试允许更多的样本运行更可靠, 最大限度地减少用户间的变异性和实际操作时间, 并产生一致的可重现的结果。因此, 建立了一个自动化版本的化验。为了证明这种检测的自动化染色, 检查了剪接变异 METΔ14的检测。这种变异是在前基因剪接过程中被跳过的致癌中外显子14的结果, 导致了受体8,9的本构活化和转化。为了具体检测 METΔ14变体, 设计了两个外显子连接探头: 一个横跨外显子13和 15 (E13/E15) 的连接点, 以检测 METΔ14变体记录, 另一条跨越外显子14和 15 (E14/E15) 的连接以检测所满足的成绩单 (图 6A)。两个探针在 2 FFPE 被准备的细胞线被测试了: H596, 表达 METΔ14变异, 并且 A549, 表达重量遇见的基因。两个探针都显示互斥表达式模式, E13/E15 探头只检测 H596 细胞中的信号, E14/E15 探头只检测 A549 细胞中的信号 (图 6B和6C)。最后, dapB 探头没有显示任何信号, 表明没有背景信号 (图 6B和6C)。总的来说, 这一数据表明, 使用自动 BaseScope 检测的 METΔ14的具体检测。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58097/58097fig1.jpg"> 图 1: 化验工作流程.该工作流包括4个主要步骤: permeabilize 细胞或组织的预处理, 探针杂交到目标 RNA, 信号放大, 以及在 brightfield 或荧光显微镜下可视化的信号检测。单个点可以使用数字图像分析平台进行量化。<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/58097/58097fig1large.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/58097/58097fig2.jpg"> 图 2: 手工化验协议的例证.整个化验可以在9小时内完成. 预处理时间可能因组织类型而异, 因此建议在使用者手册中查阅附录 A, 以了解有关潜伏期的组织预处理建议。<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/58097/58097fig2large.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/58097/58097fig3.jpg"> 图 3: 具有代表性的外显子连接检测图像.(A) 这里显示的是 egfr 和 EGFRvIII 记录的外显子组织, 以及一个描述双 Z 外显子连接探头的示意图, 横跨交叉点以检测 egfr 或 EGFRvIII。(B) 使用 (a) 中列出的探针对两个 FFPE 胶质瘤样品进行外显子结检测, 并进行阳性对照探针、POLR2A 和阴性对照探针 dapB。<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/58097/58097fig3large.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/58097/58097fig4.jpg"> 图 4: 具有代表性的短目标序列检测图像.(A) 这里显示的是 Jurkat 细胞中的 CDR3 序列。黑色的序列是侧翼的共同序列, 红色的序列是 CDR3α或 CDR3β的唯一。针对这些序列设计了单双 Z 短目标序列探头。(B) 对以 (A) 中的序列为靶向 FFPE 细胞颗粒制备的 Jurkat 细胞进行短程靶向检测, 并将 dapB 用作阴性对照。<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/58097/58097fig4large.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/58097/58097fig5.jpg"> 图 5: 具有代表性的点突变检测图像.(A) 对 H2229 细胞 (egfr L858 的纯合性) 和 H1975 细胞 (egfr L858R 突变异型) 进行了实验, 采用单双 Z 点突变探针, 以 egfr FFPE 序列或 egfr L858 为对象制备 L858R 细胞颗粒;变异序列。(B) 对 HuT78 细胞 (KRAS G12A) 和 SW116 细胞 (异型 KRAS 突变) 进行了点突变检测, 其方法是使用单双 Z 点突变探针, 以 G12A FFPE 粒子序列或KRAS G12A 突变序列。<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/58097/58097fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/58097/58097fig6.jpg"> 图 6: 带有外显子结检测的自动染色的代表性图像.(A) 这里显示的是用于满足 METΔ14记录的外显子组织和一幅描述单双 Z 外显子连接探头的示意图, 横跨连接点以检测满足了 METΔ14。(B) 和 (C) 使用 (A) 所列探针, 以及阴性对照探针 dapB, 对 H596 细胞 (表达 METΔ14) 和 A549 细胞 (表达满足度) 进行了自动外显子结检测。<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/58097/58097fig6large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>外显子连接</td>
			<td>短序列</td>
			<td>点突变</td>
		</tr>
<tr>
<td>拼接变形/异构体</td>
			<td>序列介于50和 300 nt 之间</td>
			<td>点突变</td>
		</tr>
<tr>
<td>圆形 RNA (circRNA)</td>
			<td>高度同源序列</td>
			<td>短 indel</td>
		</tr>
<tr>
<td>基因融合</td>
			<td>TCR 克隆的 CDR3 序列</td>
			<td>基因编辑</td>
		</tr>
<tr>
<td>基因击倒 (高)</td>
			<td>预 miRNA</td>
			<td></td>
		</tr>
<tr>
<td></td>
			<td>小核仁 RNA (snoRNA)</td>
			<td></td>
		</tr>
<tr>
<td></td>
			<td>基因编辑</td>
			<td></td>
		</tr>
</tbody></table>表 1: 应用就地化验.该检测的应用主要有3类: 外显子结、短靶序列和点突变。每个列中列出的是每个类别的特定应用程序示例。

Watch this Scientific Journal Video about Visualizing Genetic Variants, Short Targets, and Point Mutations in the Morphological Tissue Context with an RNA In Situ Hybridization Assay at JoVE.com

Visualizing Genetic Variants, Short Targets, and Point Mutations in the Morphological Tissue Context with an RNA In Situ Hybridization Assay

在这里, 我们描述了原位杂交检测, 使敏感和特定的检测序列短于50核苷酸的单核苷酸分辨率在单细胞水平。该检测可以手动或自动执行, 可以在组织上下文中实现拼接变体、短序列和突变的可视化。

用 RNA原位杂交法对形态学组织环境中的遗传变异、短靶点和突变进行可视化

Because precision medicine is highly dependent on the accurate detection of biomarkers, there is an increasing need for standardized and robust ...

visualizing-genetic-variants-short-targets-point-mutations

Universidad San Sebastian

Research

JoVE Journal

Biology

Visualizing Genetic Variants, Short Targets, and Point Mutations in the Morphological Tissue Context with an RNA In Situ Hybridization Assay

10.5K 次观看.  Advanced Cell Diagnostics, Inc. 在这里, 我们描述了原位杂交检测, 使敏感和特定的检测序列短于50核苷酸的单核苷酸分辨率在单细胞水平。该检测可以手动或自动执行, 可以在组织上下文中实现拼接变体、短序列和突变的可视化。

视频: Visualizing Genetic Variants, Short Targets, and Point Mutations in the Morphological Tissue Context with an RNA In Situ Hybridization Assay

High Resolution Fluorescent In Situ Hybridization in Drosophila Embryos and Tissues Using Tyramide Signal Amplification

In our efforts to determine the patterns of expression and subcellular localization of Drosophila RNAs on a genome-wide basis, and in a variety of tissues, we have developed numerous modifications and improvements to our original fluorescent in situ hybridization (FISH) protocol. To facilitate throughput and cost effectiveness, all steps, from probe generation to signal detection, are performed using exon 96-well microtiter plates. Digoxygenin (DIG)-labelled antisense RNA probes are produced using either cDNA clones or genomic DNA as templates. After tissue fixation and permeabilization, probes are hybridized to transcripts of interest and then detected using a succession of anti-DIG antibody conjugated to biotin, streptavidin conjugated to horseradish peroxidase (HRP) and fluorescently conjugated tyramide, which in the presence of HRP, produces a highly reactive intermediate that binds to electron dense regions of immediately adjacent proteins. These amplification and localization steps produce a robust and highly localized signal that facilitates both cellular and subcellular transcript localization. The protocols provided have been optimized to produce highly specific signals in a variety of tissues and developmental stages. References are also provided for additional variations that allow the simultaneous detection of multiple transcripts, or transcripts and proteins, at the same time.

High Resolution Fluorescent In Situ Hybridization in Drosophila Embryos and Tissues Using Tyramide Signal Amplification

The described RNA in situ hybridization protocol allows the detection of RNA in whole Drosophila embryos or dissected tissues. Using 96-well microtiter plates and tyramide signal amplification, transcripts can be detected at high resolution, sensitivity, and throughput, and at a relatively low cost.

高分辨率荧光原位杂交在果蝇胚胎和组织中使用 Tyramide 信号放大

In our efforts to determine the patterns of expression and subcellular localization of Drosophila RNAs on a genome-wide basis, and in a variety of ...

科研

遗传学

Spatial Profiling of Protein and RNA Expression in Tissue: An Approach to Fine-Tune Virtual Microdissection

Multiplexing enables the assessment of several markers on the same tissue while providing spatial context. Spatial Omics technologies allow both protein and RNA multiplexing by leveraging photo-cleavable oligo-tagged antibodies and probes, respectively. Oligos are cleaved and quantified from specific regions across the tissue to elucidate the underlying biology. Here, the study demonstrates that automated custom antibody visualization protocols can be utilized to guide ROI selection in conjunction with spatial proteomics assays. This specific method did not show acceptable performance with spatial transcriptomics assays. The protocol describes the development of a 3-plex immunofluorescent (IF) assay for marker visualization on an automated platform, using tyramide signal amplification (TSA) to amplify the fluorescent signal from a given protein target and increase the antibody pool to choose from. The visualization protocol was automated using a thoroughly validated 3-plex assay to ensure quality and reproducibility. In addition, the exchange of DAPI for SYTO dyes was evaluated to allow imaging of TSA-based IF assays on the spatial profiling platform. Additionally, we tested the ability of selecting small ROIs using the spatial transcriptomics assay to allow the investigation of highly-specific areas of interest (e.g., areas enriched for a given cell type). ROIs of 50 &#181;m and 300 &#181;m diameter were collected, which corresponds to approximately 15 cells and 100 cells, respectively. Samples were made into libraries and sequenced to investigate the capability to detect signals from small ROIs and profile-specific regions of the tissue. We determined that spatial proteomics technologies highly benefit from automated, standardized protocols to guide ROI selection. While this automated visualization protocol was not compatible with spatial transcriptomics assays, we were able to test and confirm that specific cell populations can successfully be detected even in small ROIs with the standard manual visualization protocol.

Here, we describe a protocol for fine-tuning regions of interest (ROIs) for Spatial Omics technologies to better characterize the tumor microenvironment and identify specific cell populations. For proteomics assays, automated customized protocols can guide ROI selection, while transcriptomics assays can be fine-tuned utilizing ROIs as small as 50 &#181;m.

组织中蛋白质和RNA表达的空间分析:一种微调虚拟显微切割的方法

Multiplexing enables the assessment of several markers on the same tissue while providing spatial context. Spatial Omics technologies allow both protein ...

癌症研究

Single Cell Analysis Of Transcriptionally Active Alleles By Single Molecule FISH

Gene transcription is an essential process in cell biology, and allows cells to interpret and respond to internal and external cues. Traditional bulk population methods (Northern blot, PCR, and RNAseq) that measure mRNA levels lack the ability to provide information on cell-to-cell variation in responses. Precise single cell and allelic visualization and quantification is possible via single molecule RNA fluorescence in situ hybridization (smFISH). RNA-FISH is performed by hybridizing target RNAs with labeled oligonucleotide probes. These can be imaged in medium/high throughput modalities, and, through image analysis pipelines, provide quantitative data on both mature and nascent RNAs, all at the single cell level. The fixation, permeabilization, hybridization and imaging steps have been optimized in the lab over many years using the model system described herein, which results in successful and robust single cell analysis of smFISH labeling. The main goal with sample preparation and processing is to produce high quality images characterized by a high signal-to-noise ratio to reduce false positives and provide data that are more accurate. Here, we present a protocol describing the pipeline from sample preparation to data analysis in conjunction with suggestions and optimization steps to tailor to specific samples.&#160;

Single molecule RNA fluorescence in situ hybridization (smFISH) is a method to accurately quantify levels and localization of specific RNAs at the single cell level. Here, we report our validated lab protocols for wet-bench processing, imaging and image analysis for single cell quantification of specific RNAs.

单分子鱼的转录活性等位基因单细胞分析

Gene transcription is an essential process in cell biology, and allows cells to interpret and respond to internal and external cues. Traditional bulk ...

生物化学

FISH 在人骨肉瘤细胞中长链非编码 RNA 定位

RNA Fluorescence In Situ Hybridization for Long Non-Coding RNA Localization in Human Osteosarcoma Cells

The important roles of long non-coding RNAs (lncRNAs) in cancer have been studied, such as regulating the proliferation, epithelial-mesenchymal transition (EMT), migration, infiltration, and autophagy of cancer cells. Localization detection of lncRNAs in cells can provide insight into their functions. By designing the lncRNA-specific antisense chain sequence followed by labeling with fluorescent dyes, RNA fluorescence in situ hybridization (FISH) can be applied to detect the cellular localization of lncRNAs. Together with the development of microscopy, the RNA FISH techniques now even allow for visualization of the poorly expressed lncRNAs. This method can not only detect the localization of lncRNAs alone, but also detect the colocalization of other RNAs, DNA, or proteins by using double-color or multicolor immunofluorescence. Here, we have included the detailed experimental operation procedure and precautions of RNA FISH by using lncRNA small nucleolar RNA host gene 6 (SNHG6) in human osteosarcoma cells (143B) as an example, to provide a reference for researchers who want to perform RNA FISH experiments, especially lncRNA FISH.

作者聚焦：用于在骨肉瘤细胞中定位 lncRNA-SNHG6 的 RNA FISH 

The present protocol describes a method of RNA fluorescence in situ hybridization to localize the lncRNAs in human osteosarcoma cells.

RNA荧光 原 位杂交在人骨肉瘤细胞中长链非编码RNA定位

The important roles of long non-coding RNAs (lncRNAs) in cancer have been studied, such as regulating the proliferation, epithelial-mesenchymal transition ...

Detection of Axonally Localized mRNAs in Brain Sections Using High-Resolution In Situ Hybridization

mRNAs are frequently localized to vertebrate axons and their local translation is required for axon pathfinding or branching during development and for maintenance, repair or neurodegeneration in postdevelopmental periods. High throughput analyses have recently revealed that axons have a more dynamic and complex transcriptome than previously expected. These analysis, however have been mostly done in cultured neurons where axons can be isolated from the somato-dendritic compartments. It is virtually impossible to achieve such isolation in whole tissues in vivo. Thus, in order to verify the recruitment of mRNAs and their functional relevance in a whole animal, transcriptome analyses should ideally be combined with techniques that allow the visualization of mRNAs in situ. Recently, novel ISH technologies that detect RNAs at a single-molecule level have been developed. This is especially important when analyzing the subcellular localization of mRNA, since localized RNAs are typically found at low levels. Here we describe two protocols for the detection of axonally-localized mRNAs using a novel ultrasensitive RNA ISH technology. We have combined RNAscope ISH with axonal counterstain using fluorescence immunohistochemistry or histological dyes to verify the recruitment of Atf4 mRNA to axons in vivo in the mature mouse and human brains.

Detection of Axonally Localized mRNAs in Brain Sections Using High-Resolution In Situ Hybridization

RNA in situ hybridization (ISH) enables the visualization of RNAs in cells and tissues. Here we show how combination of RNAscope ISH with immunohistochemistry or histological dyes can be successfully used to detect mRNAs localized to axons in sections of mouse and human brains.

在脑切片检测轴突本地化mRNAs的高空间分辨率<em&gt;原位</em&gt;杂交

mRNAs are frequently localized to vertebrate axons and their local translation is required for axon pathfinding or branching during development and for ...

神经科学

In Situ Hybridization for the Precise Localization of Transcripts in Plants

With the advances in genomics research of the past decade, plant biology has seen numerous studies presenting large-scale quantitative analyses of gene expression. Microarray and next generation sequencing approaches are being used to investigate developmental, physiological and stress response processes, dissect epigenetic and small RNA pathways, and build large gene regulatory networks1-3. While these techniques facilitate the simultaneous analysis of large gene sets, they typically provide a very limited spatiotemporal resolution of gene expression changes. This limitation can be partially overcome by using either profiling method in conjunction with lasermicrodissection or fluorescence-activated cell sorting4-7. However, to fully understand the biological role of a gene, knowledge of its spatiotemporal pattern of expression at a cellular resolution is essential. Particularly, when studying development or the effects of environmental stimuli and mutants can the detailed analysis of a gene's expression pattern become essential. For instance, subtle quantitative differences in the expression levels of key regulatory genes can lead to dramatic phenotypes when associated with the loss or gain of expression in specific cell types. 
Several methods are routinely used for the detailed examination of gene expression patterns. One is through analysis of transgenic reporter lines. Such analysis can, however, become time-consuming when analyzing multiple genes or working in plants recalcitrant to transformation. Moreover, an independent validation to ensure that the transgene expression pattern mimics that of the endogenous gene is typically required. Immunohistochemical protein localization or mRNA in situ hybridization present relatively fast alternatives for the direct visualization of gene expression within cells and tissues. The latter has the distinct advantage that it can be readily used on any gene of interest. In situ hybridization allows detection of target mRNAs in cells by hybridization with a labeled anti-sense RNA probe obtained by in vitro transcription of the gene of interest. 
Here we outline a protocol for the in situ localization of gene expression in plants that is highly sensitivity and specific. It is optimized for use with paraformaldehyde fixed, paraffin-embedded sections, which give excellent preservation of histology, and DIG-labeled probes that are visualized by immuno-detection and alkaline-phosphatase colorimetric reaction. This protocol has been successfully applied to a number of tissues from a wide range of plant species, and can be used to analyze expression of mRNAs as well as small RNAs8-14.

In Situ Hybridization for the Precise Localization of Transcripts in Plants

The in situ hybridization protocol described here allows a direct localization of mRNA and small RNA expression at the cellular level with high sensitivity and specificity. The procedure is optimized for paraffin-embedded plant tissue sections, is applicable to a wide range of plants and tissues, and can be completed within ten days.

原位杂交技术在植物中的精确定位成绩单

With the advances in genomics research of the past decade, plant biology has seen numerous studies presenting large-scale quantitative analyses of gene ...

生物学

Radioactive in situ Hybridization for Detecting Diverse Gene Expression Patterns in Tissue

Knowing the timing, level, cellular localization, and cell type that a gene is expressed in contributes to our understanding of the function of the gene. Each of these features can be accomplished with in situ hybridization to mRNAs within cells. Here we present a radioactive in situ hybridization method modified from Clayton et al. (1988)1 that has been working successfully in our lab for many years, especially for adult vertebrate brains2-5. The long complementary RNA (cRNA) probes to the target sequence allows for detection of low abundance transcripts6,7. Incorporation of radioactive nucleotides into the cRNA probes allows for further detection sensitivity of low abundance transcripts and quantitative analyses, either by light sensitive x-ray film or emulsion coated over the tissue. These detection methods provide a long-term record of target gene expression. Compared with non-radioactive probe methods, such as DIG-labeling, the radioactive probe hybridization method does not require multiple amplification steps using HRP-antibodies and/or TSA kit to detect low abundance transcripts. Therefore, this method provides a linear relation between signal intensity and targeted mRNA amounts for quantitative analysis. It allows processing 100-200 slides simultaneously. It works well for different developmental stages of embryos. Most developmental studies of gene expression use whole embryos and non-radioactive approaches8,9, in part because embryonic tissue is more fragile than adult tissue, with less cohesion between cells, making it difficult to see boundaries between cell populations with tissue sections. In contrast, our radioactive approach, due to the larger range of sensitivity, is able to obtain higher contrast in resolution of gene expression between tissue regions, making it easier to see boundaries between populations. Using this method, researchers could reveal the possible significance of a newly identified gene, and further predict the function of the gene of interest.

Radioactive in situ Hybridization for Detecting Diverse Gene Expression Patterns in Tissue

This protocol is successfully used to quantitatively detect levels and spatial patterns of mRNA expression in multiple tissue types across vertebrate species. The method can detect low abundance transcripts and allows processing of hundreds of slides simultaneously. We present this protocol using expression profiling of avian embryonic brain formation as an example.

放射性的原位杂交检测组织不同的基因表达模式

Knowing the timing, level, cellular localization, and cell type that a gene is expressed in contributes to our understanding of the function of the gene. ...

Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos

Assessing the expression pattern of a gene, as well as the subcellular localization properties of its transcribed RNA, are key features for understanding its biological function during development. RNA in situ hybridization (RNA-ISH) is a powerful method used for visualizing RNA distribution properties, be it at the organismal, cellular or subcellular levels 1. RNA-ISH is based on the hybridization of a labeled nucleic acid probe (e.g. antisense RNA, oligonucleotides) complementary to the sequence of an mRNA or a non-coding RNA target of interest 2. As the procedure requires primary sequence information alone to generate sequence-specific probes, it can be universally applied to a broad range of organisms and tissue specimens 3. Indeed, a number of large-scale ISH studies have been implemented to document gene expression and RNA localization dynamics in various model organisms, which has led to the establishment of important community resources 4-11. While a variety of probe labeling and detection strategies have been developed over the years, the combined usage of fluorescently-labeled detection reagents and enzymatic signal amplification steps offer significant enhancements in the sensitivity and resolution of the procedure 12. Here, we describe an optimized fluorescent in situ hybridization method (FISH) employing tyramide signal amplification (TSA) to visualize RNA expression and localization dynamics in staged Drosophila embryos. The procedure is carried out in 96-well PCR plate format, which greatly facilitates the simultaneous processing of large numbers of samples.

Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos

Here we describe a whole-mount fluorescent in situ hybridization (FISH) protocol for determining the expression and localization properties of RNAs expressed during embryogenesis in the fruit fly, Drosophila melanogaster.

用 RNA原位杂交法对形态学组织环境中的遗传变异、短靶点和突变进行可视化

摘要

探索更多视频

此视频中的章节

高分辨率荧光原位杂交在果蝇胚胎和组织中使用 Tyramide 信号放大

组织中蛋白质和RNA表达的空间分析:一种微调虚拟显微切割的方法

单分子鱼的转录活性等位基因单细胞分析

RNA荧光原位杂交在人骨肉瘤细胞中长链非编码RNA定位

RNA荧光原位杂交在人骨肉瘤细胞中长链非编码RNA定位

RNA荧光原位杂交在人骨肉瘤细胞中长链非编码RNA定位

在脑切片检测轴突本地化mRNAs的高空间分辨率

原位杂交技术在植物中的精确定位成绩单

放射性的原位杂交检测组织不同的基因表达模式

全山RNA荧光

用 RNA原位杂交法对形态学组织环境中的遗传变异、短靶点和突变进行可视化

摘要

探索更多视频

此视频中的章节

高分辨率荧光原位杂交在果蝇胚胎和组织中使用 Tyramide 信号放大

组织中蛋白质和RNA表达的空间分析:一种微调虚拟显微切割的方法

单分子鱼的转录活性等位基因单细胞分析

RNA荧光 原 位杂交在人骨肉瘤细胞中长链非编码RNA定位

RNA荧光 原 位杂交在人骨肉瘤细胞中长链非编码RNA定位

RNA荧光 原 位杂交在人骨肉瘤细胞中长链非编码RNA定位

在脑切片检测轴突本地化mRNAs的高空间分辨率

原位杂交技术在植物中的精确定位成绩单

放射性的原位杂交检测组织不同的基因表达模式

全山RNA荧光

RNA荧光原位杂交在人骨肉瘤细胞中长链非编码RNA定位

RNA荧光原位杂交在人骨肉瘤细胞中长链非编码RNA定位

RNA荧光原位杂交在人骨肉瘤细胞中长链非编码RNA定位