MAM、FS 和米高梅由 NIEHS（项目 4、P42ES027704、PI、Rusyn）资助。MAM、FS、RMM、米高梅和PKS由CPRIT资助的GCC高级显微镜和图像信息学中心（RP170719）提供支持。贝勒医学院的综合显微镜核心也提供成像支持，资金来自NIH（DK56338和CA125123）、CPRIT（RP150578）、丹·邓肯综合癌症中心和约翰·邓恩湾海岸化学基因组学联合会。

为了确保最佳效果，本协议的湿实验室部分要求所有步骤都提供标准的无 RNase 预防措施。例如，非常鼓励使用过滤的移液器尖端、无菌容器和无 RNase 缓冲器。还建议使用 RNase 抑制剂，如瓦纳迪尔核糖核苷复合物，特别是对于低丰度目标RNA。
1. 细胞培养和实验设置
<ol><li>保持粘附的MCF-7乳腺癌细胞（ATCC #HTB-22）在T75组织培养瓶与苯酚红色免费（只需要激素刺激实验）Dulbecco的改良鹰的介质（DMEM）补充10%胎儿牛血清（FBS），2mM L-谷氨酰胺， 1 nM 丙烯酸钠、50 I.U./mL 青霉素和 50 微克/毫升链霉素。</li>
	<li>将酸蚀刻、多D-赖氨酸涂层的圆形盖片（0.16至0.19毫米厚，12毫米直径）放入24个多井组织培养板中。 注:使用1 M HCl酸蚀盖唇和涂层与1毫克/mL溶液的多D-赖氨酸重组无菌PBS（磷酸盐缓冲盐水没有Ca+ 和Mg+）根据标准协议。</li>
	<li>从细胞中取出生长介质，用5mL的无菌PBS（磷酸盐缓冲盐水，无Ca+ 和Mg+）洗一次。使用 2-3 mL 的 Trypsin-EDTA 0.25% 溶液分离 MCF-7 电池。一旦细胞分离，用同等数量的生长介质中和Trypsin-EDTA 0.25%的溶液。</li>
	<li>将介质悬浮中的细胞转移到 15 mL 锥形管中，以 391 x g 的速度旋转 1 分钟，以颗粒出细胞。小心地将介质从锥形管中取出，而不会干扰细胞颗粒，并在适量的治疗介质中重新使用细胞。 注:治疗介质为苯酚-红色免费DMEM，辅以5%的木炭-德克斯特兰剥离和透析FBS，2mM L-谷氨酰胺，1nM丙状酸钠，50 I.U./mL青霉素和50μg/mL链霉素。这种介质是类固醇激素刺激实验的关键， 因为血清成分在很大程度上耗尽类固醇通过木炭剥离介质， 并通过透析减少生长因子.</li>
	<li>在 500 μL 的治疗介质中，以每口 60，000 到 70，000 个细胞在 24 个多井板的覆盖唇上播种。对于这个实验，细胞汇合应该是70-80%左右的治疗启动。根据细胞类型、生长速度和实验长度优化细胞播种密度，以避免汇合，从而使图像分析复杂化。 注意:为了进行最佳的成像和图像分析，请避免细胞堵塞。为此，在分配到井中之前，通过抽水（或短暂涡流）将细胞的无名小马彻底混合。在将镀细胞放回孵化器之前，让细胞在组织培养罩中沉淀约20分钟有助于减少细胞团块。</li>
	<li>在治疗前至少两天孵育细胞，以确保细胞周期同步和背景减少，因为任何信号分子留在生长介质的剥离/透析血清中。</li>
	<li>在治疗介质中孵育48小时后，用10nM 17+-雌二和车辆控制（DMSO）治疗MCF-7细胞，在治疗介质中稀释24小时。这种治疗使用饱和剂量的E2来诱导最大的反应。执行时间过程和剂量反应实验，以经验确定不同目标基因、细胞模型和治疗类型的条件。例如，请参阅我们最近的出版物7。</li>
</ol>2. 准备缓冲器
<ol><li>要准备固定缓冲，稀释纯化的单体甲醛（由电子显微镜供应商以 16% 的副甲醛形式出售）到 4% 的无菌 PBS Ca+/Mg+ （磷酸盐缓冲盐水加 Ca+ 和 Mg++）。将瓦纳迪尔核糖核酸复合物 （VRC） 添加到固定缓冲器中，最终浓度为 2 mM，以延迟 RNA 降解。<ol>
<li>根据 24 个多井板每口井需要 300-500 μL 的固定性，计算固定缓冲的总金额。将 4% 甲醛储存在冰上，直到固定步骤中需要。 注:为每个实验使固定缓冲器新鲜。 警告:甲醛是一种通过皮肤吸收的畸胎原。根据您的机构法规，将这种化学物质与适当的 PPE 一起使用烟雾罩中。</li>
	</ol>
</li>
	<li>对于渗透步骤，在无核酸水中准备 70% 乙醇。另一种选择是 0.5% 特里顿 -X100 无菌 PBS Ca+/Mg++ 加上 2 mM 的 VRC。使用每口井 500 微升缓冲器计算所需的渗透缓冲总量。</li>
	<li>要准备9 mL的杂交缓冲器，加入2 mL的库存50%脱硫酸盐和1mL的20倍SSC（盐酸钠柠檬酸盐）缓冲到6mL的核酸酶免费水。漩涡彻底混合杂交缓冲器。此缓冲器可在 4 °C 下存储长达一周。</li>
	<li>有五个单独的洗涤步骤，需要2倍盐酸钠（SSC）缓冲。通过预测每个洗涤步骤每个盖唇的缓冲器 500 μL 来计算所需的 2 倍 SSC 缓冲器的最终体积。在无核酸酶水中稀释所需的库存量 20 倍 SSC 缓冲。2 mM VRC 可以添加到洗涤步骤作为额外的预防措施。此缓冲器可在处理步骤期间在室温下存储。</li>
</ol>3. 固定RNA鱼
<ol><li>治疗后，将介质从井中取出，用无菌、冷的PBSCa +/Mg+洗一次。如果使用低粘度细胞模型，请不要使用真空吸气液:使用手动管道小心地从井边移除介质，并省略初始洗涤步骤。在冰上添加 500 μL 固定缓冲区 30 分钟。 注意:如果样品容易发生RNA降解，请此时使用具有 2 mM VRC 的无菌 PBS 进行洗涤步骤。</li>
	<li>按照制度规定，清除固定物并将其处理在化学废物中。用冷无菌PBS Ca+/Mg++清洗细胞两次，洗3分钟。</li>
	<li>取出PBS，在每口井中加入500微升70%乙醇。用石蜡塑料薄膜密封24井板。在 4 °C 的旋转器上放置至少四个小时。最好将样品留在 70% 乙醇中过夜。在此点上可以暂停协议，样品可以储存长达一周的 70% 乙醇。 注:PBS 中具有 2 mM VRC 解决方案的 0.5% Triton-X100 可用作替代方案。在旋转器的室温下，将样品在这种渗透缓冲器的 500 μL 中孵育 20 分钟。如果使用此渗透缓冲，则此时不能暂停协议，并且必须继续通过杂交步骤。</li>
</ol>4. RNA鱼的杂交
<ol><li>取出渗透缓冲器，在 500 μL 的 2 倍 SSC 缓冲器外加 10% 的甲酰胺中清洗一次，在室温下旋转 5 分钟。</li>
	<li>准备完整的杂交缓冲。计算每个覆盖唇大约 30 μL 的完整杂交缓冲。将外酰胺添加到杂交缓冲器中，以达到所需的百分比。例如，如果需要 500 μL 的完整杂交缓冲，则由 450 μL 的先前制造的杂交缓冲器加上 50 μL 的分子级甲酰胺组成，达到 10% vol/vol。漩涡短暂混合。 警告:甲酰胺是一种通过皮肤吸收的畸胎原。根据机构规定，将这种化学物质与适当的 PPE 一起用于烟气罩中。</li>
	<li>稀释GREB1 intron（标有Atto 647N）和GREB1外孔（标有类星体570）探针1:300在杂交缓冲。通过管道混合。保护此缓冲器免受光线照射并立即使用。 注:这些探测器的设计和制造详细在斯托西等人。探针通常作为干燥的寡核苷酸探针池提供，必须根据制造商协议14在无RNASeTE缓冲器中重建。因此，这些探头的库存溶液为 12.5 μM;探头的工作浓度为 42 nM。</li>
	<li>为隔夜杂交步骤准备一个加湿室。放下一块石蜡塑料薄膜，未暴露的一面向上，放入玻璃Petri盘中，用表面净化剂清洗，以去除鼻塞。用无菌水饱和两条纸巾，并沿着培养皿的边缘放置，以提供湿度，因为干燥会破坏特定的标签。</li>
	<li>通过在杂交缓冲器中将 30 μL 的探针点入石蜡塑料薄膜的干净侧面，从而对探针进行引线。分发探针的引号，使盖片不会相互接触。</li>
	<li>使用消毒钳，轻轻地将盖唇细胞侧向下翻转到玻璃培养皿中的探针上。避免气泡，不要对盖片施加压力。 注意:不要丢弃带有 2 倍 SSC 缓冲器的组织培养板，因为后续步骤需要这种培养板。</li>
	<li>用石蜡塑料薄膜密封玻璃培养皿，用铝箔盖住盘子，以防止光线暴露在荧光素中。在平面非旋转表面的 37 °C 下，在长达 16 小时的夜间孵育。孵化时间可以经验上有所不同，但建议至少4小时。 注:在杂交缓冲中孵育时，盖唇容易滑动，这可能导致覆盖唇上的干点并损坏样品。</li>
</ol>5. 准备成像样品 
<ol><li>第二天，用钳子从加湿室中取出盖片，然后用细胞侧向上返回24井板。加入 500 μL 的新鲜 2x SSC 缓冲器加上 10% 的表单酰胺，以洗掉多余的探头和非特异性杂交。 注意:从此开始保护样品免受光线照射。</li>
	<li>在加热旋转器上用 500 μL 的 2 倍 SSC 缓冲器加上 10% 的甲酰胺清洗细胞两次，每次 37°C 时清洗 15 分钟。</li>
	<li>在旋转器上的室温下，在 2 倍 SSC 缓冲器中，以 1 μg/mL 的 1 微克/mL 与 DAPI 对抗 DNA，持续 10 分钟。</li>
	<li>在室温下用 2 倍 SSC 缓冲器洗一次，5 分钟。</li>
	<li>使用非硬化安装介质将盖片安装到玻璃滑梯上，然后用纸巾去除多余的 2 倍 SSC 缓冲器，并在无核糖水中快速清洗，以消除多余的盐。最后，用清晰的指甲油密封盖片。</li>
</ol>6. 高分辨率显微镜和图像处理
<ol><li>使用 60 倍或 100 倍的机油目标和 sCMOS 相机在广域表观显微镜上对样品进行成像。查看用于此实验的特定显微镜和目标的材料表。<ol>
<li>一旦样品得到充分处理，就完成成像，以避免信号因时间而退化。 注:本实验中使用了折射指数 （RI） 为 1.516 的浸入油。应进行经验测试，将油 RI 与特定样品匹配，因为 RI 不匹配将导致点扩散函数的偏差，而点扩散函数将影响图像去革命。</li>
	</ol>
</li>
	<li>图像的横向像素大小为 0.10827 μm。</li>
	<li>使用预期强度最高的样品（即正控件），优化来自光源的照明量（即百分比传输）和每个通道的曝光时间（DAPI、TRITC、CY5 滤光片）。在本次实验中，E2 处理过的样品预计将对两组 GREB1 探针具有更高的强度。一般来说，GREB1 intron 和 Exon 探头的传输百分比为 50 % - 100%，曝光时间范围为 0.25 - 0.60 秒。</li>
	<li>此外，在避免光模糊和/或相机像素饱和的条件下获取图像。根据我们的经验，背景和信号强度之间应有最低 ±10 倍的差异。由于样品中的非特定信号（即覆盖唇上的污垢、细胞外的探针聚合物），可能会发生很少的像素/字段饱和，这是可以接受的，但前提是饱和区域不包括在量化中。</li>
	<li>要设置 z 堆栈的采集，则关注 DAPI 通道中的示例。选择 Z 堆栈的顶部和底部，在 DAPI 染色核失去焦点的距离。我们使用的 z 堆栈步进大小为每片 0.25 μm，整个 z 堆栈应跨越整个单元格（MCF-7 单元中约 10 μm）。但是，z 堆栈步进大小将根据所使用的目标而更改，并且应遵循奈奎斯特采样标准。 注:Introns 通常只存在于原子核中:然而，外在体都在细胞核和细胞质中。因此，设置上述 z 堆栈将捕获大部分的内电和外向标记，因为 z 堆栈包含整个单元格。</li>
	<li>通常，在初步实验中至少映像 200 个细胞进行定量分析，以捕获细胞之间响应和变异程度的快照。</li>
	<li>某些成像细胞被排除在最终分析之外（即辍学率），因为它们将被分析管道（即接触图像边界的细胞、凋形/吸血细胞）过滤掉。 注意:在选择图像区域时，需要注意几个因素。选择由 DAPI 标记的核荧光定义的均匀分布、非重叠细胞的区域。此外，尝试选择沿图像区域边缘核数最少的区域，以避免计算部分细胞。对于需要统计分析的实验，自动、公正的成像总是首选。</li>
	<li>获取后，使用具有攻击性的恢复算法（即迭代次数）对图像进行解压。生成图像分析的最大强度投影（如果需要特定的 3D 分析，则省略此步骤）。根据所使用的相机的位深度保存所有投影图像;在我们的情况下，16 位 TIFF 灰度图像被保存。RGB 图像的深度降低，导致信息丢失;因此，RGB 图像不适合图像分析。</li>
</ol>7. 图像分析 
<ol><li>阅读说明并下载从 github（https://github.com/pankajmath/RNA_FISH_analysis）的 jupyter 笔记本。</li>
	<li>安装 Python 阿纳康达分发（https://www.anaconda.com/distribution/） 版本 3.6 或更高。</li>
	<li>打开 anaconda 提示并键入安装命令，为 anaconda 安装简单的ITK（https://anaconda.org/SimpleITK/simpleitk）。</li>
	<li>打开阿纳康达导航器并推出朱皮特笔记本。它将打开显示目录和文件的 Web 浏览器。浏览目录结构，以到达包含从 github 下载的 jupyter 笔记本的目录。</li>
	<li>打开笔记本，通过按 移位 和同时 输入 来运行每个单元格。</li>
	<li>关注笔记本中提供的每个功能和步骤的详细信息。对于不同的图像集，可能需要更改某些参数值。 注:简言之，使用块大小为 251 像素的本地阈值从 DAPI 通道中分段核，填充孔并删除小于 100 像素的对象。触摸核使用分水岭算法进行分割。然后，核面罩被扩张了100像素，分水岭被用来分离接触物体，使用核作为盆。为了识别稳定的状态RNA（外在），使用了大松阈值:对于转录活动等位基因（intron），首先应用了高斯模糊（sigma+1），然后使用了最大熵阈值。</li>
</ol>

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作者没有什么可透露的。

所描述的 smFISH 方法基于先前发布的协议 7、12、14。在此协议中，我们解释了从湿实验室（包括播种密度、固定时间、渗透度和探针浓度）到成像和图像分析（包括种子密度、渗透度和探针浓度）优化的关键步骤，为有兴趣对基因转录进行单细胞分析的实验室提供了完整的实验管道。
为了促进单细胞分析?...

基因转录和翻译是生物学中心教条中涉及的两个主要过程，从DNA序列到RNA，再到蛋白质1。在这项研究中，我们专注于在转录过程中产生信使RNA（mRNA）。新生的RNA分子包括非编码内电和编码外子。在mRNA进入细胞质以在核糖体2、3上进行翻译之前，Introns被共同转录拼接出来。
传统上，mRNA 的测量是在细胞的批量群（数亿到数百万）中进行的，具有经典的测定方法，如 RT-qPCR 或北方印迹。虽然非常强大，但这些方法是有限的，因为它们不能提供对细胞间变异（表型异质性）、对转录活性等位基因数量的识别，以及也许更重要的是缺乏空间信息的见解。最近，允许单细胞RNA测序（RNAseq）的全基因组技术开始弥合成像方法和测序4之间的差距。但是，RNAseq 的检测效率相对较低，处理步骤导致空间信息完全丢失5。虽然单细胞RNASeq可以提供对同源细胞群中表型异质性的见解，但原位杂交（RNA-FISH）可以促进在空间水平上和在6、7等位基因基础上更全面地探索目标基因表达。
RNA FISH 是一种技术，用于检测和定位固定细胞中的目标RNA分子。与早期的艰苦方法不同，目前的最先进的RNA-FISH利用了与目标RNA序列互补的商用核酸探针，这些探针通过沃森-克里克碱基配对8杂交到目标上。早期的原位杂交技术涉及Gall和Pardue在1969年建立的类似协议，因为样品是用核酸探针处理的，核酸探针专门杂交到靶RNA9。最初，测定是使用放射性或色度检测进行的:然而，在20世纪80年代，发展荧光原位杂交（FISH）协议DNA鱼和后来的RNA鱼开辟了路径，走向更敏感的检测，并有可能复用10，11。
RNA FISH的进一步发展已导致能够检测和量化单RNA分子（smFISH）12，13。直接检测是探针本身被标记为氟磷的方法。smFISH 的一个挑战是，需要足够的杂交探头/目标，以促进荧光信号作为独特的衍射限制点的检测。为了解决这个问题，人们可以使用一组短的单链DNA寡核苷酸，补充到目标RNA8，12，14的不同区域。多个探针的结合会增加局部荧光密度，使RNA通过荧光显微镜可见为一个独特的高强度点。这种方法是有利的，因为探针组池中寡核苷酸的脱靶结合可以通过定量分析点大小和强度来区分真实信号和虚假的寡核苷酸结合，从而通过减少误报12来促进更准确的分析。
检测 mRNA 的替代方法是经典的 BAC 克隆基刻痕翻译方法和最近开发的分支 DNA 系统。在BAC克隆的刻痕转换方法中，要标记的DNA是酶刻痕，在暴露的3'端添加了一种新的核苷酸。DNA聚合酶的活性我添加了一个标记的核苷酸，导致所需的探针15，16。分支DNA技术涉及寡核苷酸探针，成对杂交到RNA目标，这些探针利用多个信号放大分子被放大。这种方法可以显著提高信号与噪声比，但放大可以扭曲精确的量化，因为荧光点在大小和强度上可能大相径庭。
作为此协议的模型系统，作为 NIEHS 超级基金研究计划的一部分，我们充分利用了该协议，以量化内分泌干扰化学物质在加尔维斯顿湾/休斯顿船舶通道的影响，我们使用雌激素受体 （ER） 阳性乳腺癌细胞系 MCF-7 与 ER 激动剂 17+ -estradiol （E2）7，18，19通宵治疗。E2调节许多ER靶点基因，包括原型ER靶基因GREB17，20，21，22。此处描述的 smFISH 协议使用一组两组针对 GREB1 的光谱分离探针集;一个杂交到外星，另一个到内电，允许测量成熟和新生的RNA7。然后，我们使用表观显微镜，结合去革命图像 smFISH 标记的样品，然后应用图像分析程序来量化每个细胞的活性等位基因和成熟RNA 的数量。

<table border="0" cellpadding="0" cellspacing="0" style="width:572px;" width="573">
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	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">17&#946; Estradiol</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">E2257</td>
			<td style="width:167px;">CAS Number 50-28-2</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Ambion SSC (20X) Buffer, Rnase-free</td>
			<td style="width:71px;">ThermoFisher Scientific</td>
			<td style="width:119px;">AM9770</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Corning DMEM with L-Glutamine and 4.5 g/L Glucose</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td style="width:119px;">MT10017CV</td>
			<td>Manufactured by Invitrogen</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">DAPI</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">D8417</td>
			<td>Without sodium pyruvate</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Dextran Sulfate, 50% Solution Sterile</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">3730-100ML</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Dulbecco&#39;s Phosphate Buffered Saline Ca++/Mg++</td>
			<td style="width:71px;">Corning</td>
			<td style="width:119px;">21030CV</td>
			<td>Manufactured by Calbiochem</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Ethanol, 200 Proof (100%)</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td style="width:119px;">07-678-005</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Falcon 24-Well Clear Flat Bottom TC-treated Multiwell Cell Culture Plate</td>
			<td style="width:71px;">Corning</td>
			<td align="right" style="width:119px;">353047</td>
			<td>Manufactured by Decon Laboratories</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Fetal Bovine Serum (FBS), 500 mL</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td style="width:119px;">50-753-2978</td>
			<td>Manufactured by Gemini Bio Products</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">GE Healthcare DeltaVision LIVE High Resolution Deconvolution Microscope</td>
			<td style="width:71px;">GE Healthcare</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Hyclone Water, Molecular Biology Grade</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td style="width:119px;">SH3053802</td>
			<td></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Immersion Oil 1.516</td>
			<td style="width:71px;">GE Healthcare Life Sciences</td>
			<td align="right" style="width:119px;">29162940</td>
			<td>Manufactured by GE Healthcare Bio-Science</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">L-glutamine Solution</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td style="width:119px;">MT25005Cl</td>
			<td>Manufactured by Corning</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">MCF-7 cells</td>
			<td style="width:71px;">ATCC</td>
			<td style="width:119px;">HTB-22</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Molecular Grade Formamide</td>
			<td style="width:71px;">Promega</td>
			<td style="width:119px;">PAH5051</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Olympus 60x/1.42 NA Objective</td>
			<td style="width:71px;">Olympus</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Parafilm</td>
			<td style="width:71px;">Bemis Company, Inc.</td>
			<td style="width:119px;">52858-076</td>
			<td>Distributed by VWR</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Paraformaldehyde, 16% Solution, EM Grade</td>
			<td style="width:71px;">Electron Microscopy Sciences</td>
			<td align="right" style="width:119px;">15710</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Penicilin-Streptomycin Solution</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td style="width:119px;">MT3001Cl</td>
			<td>Manufactured by Corning</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Ribonucleoside Vanadyl Complex</td>
			<td style="width:71px;">VWR</td>
			<td style="width:119px;">101229-716</td>
			<td>Manufactured by New England Biosciences</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">RNase Away</td>
			<td style="width:71px;">Ambion</td>
			<td align="right" style="width:119px;">9780</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Sodium pyruvate, 100 mM</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td style="width:119px;">11-360-070</td>
			<td>Manufactured by Gibco</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Thermo Scientific Glass Coverslips, #1.5</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td style="width:119px;">12-545-81P</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Triton X-100, 100 mL</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td style="width:119px;">BP151-100</td>
			<td>Manufactured by Fisher BioReagents</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Trypsin-EDTA Solution 0.25%</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">T4049-500ML</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Vectashield Antifade Mounting Medium 10 mL</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td style="width:119px;">NC9265087</td>
			<td>Manufactured by Vector Laboratories</td>
		</tr>
	</tbody>
</table>

single cell analysis of transcriptionally active alleles by single molecule fish

基因转录是细胞生物学的一个基本过程，它允许细胞解释和响应内部和外部的线索。测量 mRNA 水平的传统大宗种群方法（北方污点、PCR 和 RNAseq）缺乏提供响应中细胞间变化信息的能力。通过单分子RNA荧光就地杂交（smFISH），可以实现精确的单细胞和过敏可视化和定量。RNA-FISH 通过将目标RNA与标记的寡核苷酸探头杂交来执行。这些数据可以以中/高吞吐量方式进行成像，并通过图像分析管道提供成熟和新生的RNA的定量数据，所有这些数据均在单个单元格级别上。多年来，实验室使用本处描述的模型系统对固定、渗透、杂交和成像步骤进行了优化，从而成功、稳健地对 smFISH 标签进行单细胞分析。样品制备和处理的主要目标是生成高质量的图像，其特点是信号与噪声比高，以减少误报并提供更准确的数据。在这里，我们提出一个协议，描述从样品制备到数据分析的管道，以及针对特定样本的建议和优化步骤。

为了通过 smFISH 分析荷尔蒙反应，例如，我们选择了用于高吞吐量检测的雌激素受体 （ER） 模型，以确定在参与 NIEHS 超级基金研究计划7的背景下，在环境灾难中存在内分泌干扰化学物质。在本实验中，粘附的MCF-7乳腺癌细胞用ER激动剂17+-雌二（E2，10 nM）或车辆（DMSO）治疗24小时。与GREB1电子（图1中的红色，图1中的红色）和外波（Quasar 570，图<strong class="xfi...

Watch this Scientific Journal Video about Single Cell Analysis Of Transcriptionally Active Alleles By Single Molecule FISH at JoVE.com

Single Cell Analysis Of Transcriptionally Active Alleles By Single Molecule FISH

单分子RNA荧光就地杂交（smFISH）是一种在单细胞水平上准确量化特定RNA水平和定位的方法。在这里，我们报告我们已验证的实验室协议，用于湿板凳处理、成像和图像分析，用于特定RNA的单细胞量化。

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 ...

single-cell-analysis-transcriptionally-active-alleles-single-molecule

Research

JoVE Journal

Biochemistry

3.1K 次观看.  GCC Center for Advanced Microscopy and Image Informatics. 单分子RNA荧光就地杂交（smFISH）是一种在单细胞水平上准确量化特定RNA水平和定位的方法。在这里，我们报告我们已验证的实验室协议，用于湿板凳处理、成像和图像分析，用于特定RNA的单细胞量化。

视频: Single Cell Analysis Of Transcriptionally Active Alleles By Single Molecule FISH

Single-molecule Manipulation of G-quadruplexes by Magnetic Tweezers

Non-canonical nucleic acid secondary structure G-quadruplexes (G4) are involved in diverse cellular processes, such as DNA replication, transcription, RNA processing, and telomere elongation. During these processes, various proteins bind and resolve G4 structures to perform their function. As the function of G4 often depends on the stability of its folded structure, it is important to investigate how G4 binding proteins regulate the stability of G4. This work presents a method to manipulate single G4 molecules using magnetic tweezers, which enables studies of the regulation of G4 binding proteins on a single G4 molecule in real time. In general, this method is suitable for a wide scope of applications in studies for proteins/ligands interactions and regulations on various DNA or RNA secondary structures.

A single-molecule magnetic tweezers platform to manipulate G-quadruplexes is reported, which allows for the study of G4 stability and regulation by various proteins.

磁性镊子 quadruplexes 单分子操作

Non-canonical nucleic acid secondary structure G-quadruplexes (G4) are involved in diverse cellular processes, such as DNA replication, transcription, RNA ...

科研

生物化学

Covalent Immobilization of Proteins for the Single Molecule Force Spectroscopy

In recent years, atomic force microscopy (AFM) based single molecule force spectroscopy (SMFS) extended our understanding of molecular properties and functions. It gave us the opportunity to explore a multiplicity of biophysical mechanisms, e.g., how bacterial adhesins bind to host surface receptors in more detail. Among other factors, the success of SMFS experiments depends on the functional and native immobilization of the biomolecules of interest on solid surfaces and AFM tips. Here, we describe a straightforward protocol for the covalent coupling of proteins to silicon surfaces using silane-PEG-carboxyls and the well-established N-hydroxysuccinimid/1-ethyl-3-(3-dimethyl-aminopropyl)carbodiimid (EDC/NHS) chemistry in order to explore the interaction of pilus-1 adhesin RrgA from the Gram-positive bacterium Streptococcus pneumoniae (S. pneumoniae) with the extracellular matrix protein fibronectin (Fn). Our results show that the surface functionalization leads to a homogenous distribution of Fn on the glass surface and to an appropriate concentration of RrgA on the AFM cantilever tip, apparent by the target value of up to 20% of interaction events during SMFS measurements and revealed that RrgA binds to Fn with a mean force of 52 pN. The protocol can be adjusted to couple via site specific free thiol groups. This results in a predefined protein or molecule orientation and is suitable for other biophysical applications besides the SMFS.

This protocol describes the covalent immobilization of proteins with a heterobifunctional silane coupling agent to silicon-oxide surfaces designed for the atomic force microscopy based single molecule force spectroscopy which is exemplified by the interaction of RrgA (pilus-1 tip adhesin of S. pneumoniae) with fibronectin.

单分子力谱中蛋白质共价固定化的研究

In recent years, atomic force microscopy (AFM) based single molecule force spectroscopy (SMFS) extended our understanding of molecular properties and ...

Proteome-wide Quantification of Labeling Homogeneity at the Single Molecule Level

Cell proteomes are often characterized using electrophoresis assays, where all species of proteins in the cells are non-specifically labeled with a fluorescent dye and are spotted by a photodetector following their separation. Single molecule fluorescence imaging can provide ultrasensitive protein detection with its ability for visualizing individual fluorescent molecules. However, the application of this powerful imaging method to electrophoresis assays is hampered by the lack of ways to characterize the homogeneity of fluorescent labeling of each protein species across the proteome. Here, we developed a method to evaluate the labeling homogeneity across the proteome based on a single molecule fluorescence imaging assay. In our measurement using a HeLa cell sample, the proportion of proteins labeled with at least one dye, which we termed &#8216;labeling occupancy&#8217; (LO), was determined to range from 50% to 90%, supporting the high potential of the application of single molecule imaging to sensitive and precise proteome analysis.

Here, we present a protocol to assess the labeling homogeneity for each protein species in a complex protein sample at the single molecule level.

单分子水平标记均匀性的全蛋白质定量

Cell proteomes are often characterized using electrophoresis assays, where all species of proteins in the cells are non-specifically labeled with a ...

Conventional BODIPY Conjugates for Live-Cell Super-Resolution Microscopy and Single-Molecule Tracking

Single molecule localization microscopy (SMLM) techniques overcome the optical diffraction limit of conventional fluorescence microscopy and can resolve intracellular structures and the dynamics of biomolecules with ~20 nm precision. A prerequisite for SMLM are fluorophores that transition from a dark to a fluorescent state in order to avoid spatio-temporal overlap of their point spread functions in each of the thousands of data acquisition frames. BODIPYs are well-established dyes with numerous conjugates used in conventional microscopy. The transient formation of red-shifted BODIPY ground-state dimers (DII) results in bright single molecule emission enabling single molecule localization microscopy (SMLM). Here we present a simple but versatile protocol for SMLM with conventional BODIPY conjugates in living yeast and mammalian cells. This procedure can be used to acquire super-resolution images and to track single BODIPY-DII states to extract spatio-temporal information of BODIPY conjugates. We apply this procedure to resolve lipid droplets (LDs), fatty acids, and lysosomes in living yeast and mammalian cells at the nanoscopic length scale. Furthermore, we demonstrate the multi-color imaging capability with BODIPY dyes when used in conjunction with other fluorescent&#160;probes. Our representative results show the differential spatial distribution and mobility of BODIPY-fatty acids and neutral lipids in yeast under fed and fasted conditions. This optimized protocol for SMLM can be used with hundreds of commercially available BODIPY conjugates and is a useful resource to study biological processes at the nanoscale far beyond the applications of this work.&#160;

Conventional BODIPY conjugates can be used for live-cell single-molecule localization microscopy (SMLM) through exploitation of their transiently forming, red-shifted ground state dimers. We present an optimized SMLM protocol to track and resolve subcellular neutral lipids and fatty acids in living mammalian and yeast cells at the nanoscopic length scale.

用于活细胞超分辨率显微镜和单分子跟踪的传统 BODIPY 结合物

Single molecule localization microscopy (SMLM) techniques overcome the optical diffraction limit of conventional fluorescence microscopy and can resolve ...

An In Vitro Single-Molecule Imaging Assay for the Analysis of Cap-Dependent Translation Kinetics

Cap-dependent protein synthesis is the predominant translation pathway in eukaryotic cells. While various biochemical and genetic approaches have allowed extensive studies of cap-dependent translation and its regulation, high resolution kinetic characterization of this translation pathway is still lacking. Recently, we developed an in vitro assay to measure cap-dependent translation kinetics with single-molecule resolution. The assay is based on fluorescently labeled antibody binding to nascent epitope-tagged polypeptide. By imaging the binding and dissociation of antibodies to and from nascent peptide&#8211;ribosome&#8211;mRNA complexes, the translation progression on individual mRNAs can be tracked. Here, we present a protocol for establishing this assay, including mRNA and PEGylated slide preparations, real-time imaging of translation, and analysis of single molecule trajectories. This assay enables tracking of individual cap-dependent translation events and resolves key translation kinetics, such as initiation and elongation rates. The assay can be widely applied to distinct translation systems and should broadly benefit in vitro studies of cap-dependent translation kinetics and translational control mechanisms.

An In Vitro Single-Molecule Imaging Assay for the Analysis of Cap-Dependent Translation Kinetics

Tracking individual translation events allows for high-resolution kinetic studies of cap-dependent translation mechanisms. Here we demonstrate an in vitro single-molecule assay based on imaging interactions between fluorescently labeled antibodies and epitope-tagged nascent peptides. This method enables single-molecule characterization of initiation and peptide elongation kinetics during active in vitro cap-dependent translation.

用于分析依赖帽的翻译动力学的 体外 单分子成像测定

Cap-dependent protein synthesis is the predominant translation pathway in eukaryotic cells. While various biochemical and genetic approaches have allowed ...

Single-Molecule Dwell-Time Analysis of Restriction Endonuclease-Mediated DNA Cleavage

This novel total internal reflection fluorescence microscopy-based assay facilitates the simultaneous measurement of the length of the catalytic cycle for hundreds of individual restriction endonuclease (REase) molecules in one experiment. This assay does not require protein labeling and can be carried out with a single imaging channel. In addition, the results of multiple individual experiments can be pooled to generate well-populated dwell-time distributions. Analysis of the resulting dwell-time distributions can help elucidate the DNA cleavage mechanism by revealing the presence of kinetic steps that cannot be directly observed. Example data collected using this assay with the well-studied REase, EcoRV - a dimeric Type IIP restriction endonuclease that cleaves the palindromic sequence GAT&#8595;ATC (where &#8595; is the cut site) - are in agreement with prior studies. These results suggest that there are at least three steps in the pathway to DNA cleavage that is initiated by introducing magnesium after EcoRV binds DNA in its absence, with an average rate of 0.17 s-1 for each step.

Using quantum-dot-labeled DNA and total internal reflection fluorescence microscopy, we can investigate the reaction mechanism of restriction endonucleases while using unlabeled protein. This single-molecule technique allows for massively multiplexed observation of individual protein-DNA interactions, and data can be pooled to generate well-populated dwell-time distributions.

限制性核酸内切酶介导的 DNA 切割的单分子停留时间分析

This novel total internal reflection fluorescence microscopy-based assay facilitates the simultaneous measurement of the length of the catalytic cycle for ...

Single Molecule Fluorescence Energy Transfer Study of Ribosome Protein Synthesis

The ribosome is a large ribonucleoprotein complex that assembles proteins processively along mRNA templates. The diameter of the ribosome is approximately 20 nm to accommodate large tRNA substrates at the A-, P- and E-sites. Consequently, the ribosome dynamics are naturally de-phased quickly. Single molecule method can detect each ribosome separately and distinguish inhomogeneous populations, which is essential to reveal the complicated mechanisms of multi-component systems. We report the details of a smFRET method based on the Nikon Ti2 inverted microscope to probe the ribosome dynamics between the ribosomal protein L27 and tRNAs. The L27 is labeled at its unique Cys 53 position and reconstituted into a ribosome that is engineered to lack L27. The tRNA is labeled at its elbow region. As the tRNA moves to different locations inside the ribosome during the elongation cycle, such as pre- and post- translocation, the FRET efficiencies and dynamics exhibit differences, which have suggested multiple subpopulations. These subpopulations are not detectable by ensemble methods. The TIRF-based smFRET microscope is built on a manual or motorized inverted microscope, with home-built laser illumination. The ribosome samples are purified by ultracentrifugation, loaded into a home-built multi-channel sample cell and then illuminated via an evanescent laser field. The reflection laser spot can be used to achieve feedback control of perfect focus. The fluorescence signals are separated by a motorized filter-turret and collected by two digital CMOS cameras. The intensities are retrieved via the NIS-Elements software.

Single molecule fluorescence energy transfer is a method that tracks the tRNA dynamics during ribosomal protein synthesis. By tracking individual ribosomes, inhomogeneous populations are identified, which shed light on mechanisms. This method can be used to track biological conformational changes in general to reveal dynamic-function relationships in many other complexed biosystems. Single molecule methods can observe non-rate limiting steps and low-populated key intermediates, which are not accessible by conventional ensemble methods due to the average effect.

单分子荧光能量转移研究核糖体蛋白质合成

The ribosome is a large ribonucleoprotein complex that assembles proteins processively along mRNA templates. The diameter of the ribosome is approximately ...

Single-Molecule Imaging of EWS-FLI1 Condensates Assembling on DNA

The fusion genes resulting from chromosomal translocation have been found in many solid tumors or leukemia. EWS-FLI1, which belongs to the FUS/EWS/TAF15 (FET) family of fusion oncoproteins, is one of the most frequently involved fusion genes in Ewing sarcoma. These FET family fusion proteins typically harbor a low-complexity domain (LCD) of FET protein at their N-terminus and a DNA-binding domain (DBD) at their C-terminus. EWS-FLI1 has been confirmed to form biomolecular condensates at its target binding loci due to LCD-LCD and LCD-DBD interactions, and these condensates can recruit RNA polymerase II to enhance gene transcription. However, how these condensates are assembled at their binding sites remains unclear. Recently, a single-molecule biophysics method-DNA Curtains-was applied to visualize these assembling processes of EWS-FLI1 condensates. Here, the detailed experimental protocol and data analysis approaches are discussed for the application of DNA Curtains in studying the biomolecular condensates assembling on target DNA.

Here, we describe the use of the single-molecule imaging method, DNA Curtains, to study the biophysical mechanism of EWS-FLI1 condensates assembling on DNA.

EWS-FLI1凝聚物在DNA上组装的单分子成像

The fusion genes resulting from chromosomal translocation have been found in many solid tumors or leukemia. EWS-FLI1, which belongs to the FUS/EWS/TAF15 ...

Single-Molecule Analysis of Sf9 Purified Superprocessive Kinesin-3 Family Motors

A complex cellular environment poses challenges for single-molecule motility analysis. However, advancement in imaging techniques have improved single-molecule studies and has gained immense popularity in detecting and understanding the dynamic behavior of fluorescent-tagged molecules. Here, we describe a detailed method for&#160;in vitro single-molecule studies of kinesin-3 family motors using Total Internal Reflection Fluorescence (TIRF) microscopy. Kinesin-3 is a large family that plays critical roles in cellular and physiological functions ranging from intracellular cargo transport to cell division to development. We have shown previously that constitutively active dimeric kinesin-3 motors exhibit fast and superprocessive motility with high microtubule affinity at the single-molecule level using cell lysates prepared by expressing motor in mammalian cells. Our lab studies kinesin-3 motors and their regulatory mechanisms using cellular, biochemical and biophysical approaches, and such studies demand purified proteins at a large scale. Expression and purification of these motors using mammalian cells would be expensive and time-consuming, whereas expression in a prokaryotic expression system resulted in significantly aggregated and&#160;inactive protein. To overcome the limitations posed by bacterial purification systems and mammalian cell lysate, we have established a robust Sf9-baculovirus expression system to express and purify these motors. The kinesin-3 motors are C-terminally tagged with 3-tandem fluorescent proteins (3xmCitirine or 3xmCit) that provide enhanced signals and decreased photobleaching. In vitro single-molecule and multi-motor gliding analysis of Sf9 purified proteins demonstrate that kinesin-3 motors are fast and superprocessive akin to our previous studies using mammalian cell lysates. Other applications using these assays include detailed knowledge of oligomer conditions of motors, specific binding partners paralleling biochemical studies, and their kinetic state.

This study details purification of KIF1A(1-393LZ), a member of kinesin-3 family, using Sf9-baculovirus expression system. In vitro single-molecule and multi-motor gliding analysis of these purified motors exhibited robust motility properties comparable to motors from mammalian cell lysate. Thus, Sf9-baculovirus system is amenable to express and purify motor protein of interest.

Sf9纯化超加工驱动蛋白-3系列电机的单分子分析

A complex cellular environment poses challenges for single-molecule motility analysis. However, advancement in imaging techniques have improved ...

Single-Molecule Diffusion and Assembly on Polymer-Crowded Lipid Membranes

Cellular membranes are highly crowded environments for biomolecular reactions and signaling. Yet, most in vitro experiments probing protein interaction with lipids employ naked bilayer membranes. Such systems lack the complexities of crowding by membrane-embedded proteins and glycans and exclude the associated volume effects encountered on cellular membrane surfaces. Also, the negatively charged glass surface onto which the lipid bilayers are formed prevents the free diffusion of transmembrane biomolecules. Here, we present a well-characterized polymer-lipid membrane as a mimic for crowded lipid membranes. This protocol utilizes polyethylene glycol (PEG)-conjugated lipids as a generalized approach for incorporating crowders into the supported lipid bilayer&#160;(SLB). First, a cleaning procedure of the microscopic slides and coverslips for performing single-molecule experiments is presented. Next, methods for characterizing the PEG-SLBs and performing single-molecule experiments of the binding, diffusion, and assembly of biomolecules using single-molecule tracking and photobleaching are discussed. Finally, this protocol demonstrates how to monitor the nanopore assembly of bacterial pore-forming toxin Cytolysin A (ClyA) on crowded lipid membranes with single-molecule photobleaching analysis. MATLAB codes with example datasets are also included to perform some of the common analyses such as particle tracking, extracting diffusive behavior, and subunit counting.

Here, a protocol to perform and analyze the binding, mobility, and assembly of single molecules on artificial crowded lipid membranes using single-molecule total internal reflection fluorescence (smTIRF) microscopy is presented.

聚合物拥挤脂质膜上的单分子扩散和组装

Cellular membranes are highly crowded environments for biomolecular reactions and signaling. Yet, most in vitro experiments probing protein interaction ...