Name: method for labeling transcripts in individual escherichia coli cells for single-molecule fluorescence in situ hybridization experiments
Uploaded: 2017-12-21T13:00:00.000+00:00
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Description: Watch this Scientific Journal Video about Method for Labeling Transcripts in Individual Escherichia coli Cells for Single-molecule Fluorescence In Situ Hybridization Experiments at JoVE.com

这项工作得到了以色列科学基金会赠款 514415 (对 BSF) 和一项非名 (NSF) 赠款2016707的支持。厄尔曼教授的支持, 也承认了。

1. 探头设计
注: 本协议使用商业上可用的寡核苷酸探针已标记荧光。探针由一组与目标 mRNA 互补的特定序列组成, 每个探针被共轭到一个单一的荧光分子。或者, 可以将荧光标记附加到探测器上, 如其他地方所述5,16。
<ol>
	<li>设计 smFISH 探针使用由阿金 Raj 开发的探针设计器16算法 (Oudenaarden 实验室, 马萨诸塞技术学院);本手稿中使用的 smFISH 探针序列列在材料表中。 注意: 在一组检测信号中, 探头的最小数目是 ~ 30。确切的数字可能根据兴趣基因变化, 特别如果非常短的抄本被测量。</li>
	<li>选择适合光学设置的染料 (参见材料表)。 注: Cy3 染料或光学等效染料与低易感性到相片漂白强烈推荐。双重标记的候选人是 Cy5 或光学上等效染料 (参见材料表)。在标记 mRNA 的风暴成像中, 合适的染料具有多周期荧光发射的能力。一些 smFISH 染料可用于风暴成像 (见材料表)。要对同一细胞中的不同基因的两个 (或更多) 抄本进行图像处理, 就应该选择共轭于染料的寡核苷酸探针, 其光谱很少或没有重叠。</li>
</ol>
2. 试剂制备
注意: 重要的是要保持样品无核糖核酸, 使用无核糖核酸的消耗品, 如过滤吸管提示和管, 并保持任何工作环境清洁。为了避免目标记录的退化, 所有使用的试剂都必须是无核酸的 (见材料表) 或 diethylpyrocarbonate (DEPC) 治疗和灭菌。
<ol>
	<li>smFISH 的缓冲区
	<ol>
		<li>准备无核糖核酸的 1x PBS (磷酸盐缓冲盐水, 溶液的磷酸盐缓冲浓度为0.01 米, 氯化钠浓度为0.154 米, (pH 7.4))。稀释无核糖核酸的 10x PBS (参见材料表) 在无核酸的水中 (见材料表) 以 1:10 v/v 比。<ol>
			<li>或者准备 DEPC 处理的 1x PBS。</li>
		</ol>
		</li>
		<li>准备无核糖核酸的 2x SSC (生理盐水-柠檬酸钠缓冲液, 0.3 米氯化钠在0.03 米柠檬酸钠, (pH 7.0))。稀释无核糖核酸的 20x SSC (见材料表) 在核酸的水中以 1:10 v/v 的比例。<ol>
			<li>另外, 准备 DEPC 处理的 2x SSC。</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>寡核苷酸探针库存解决方案。
	<ol>
		<li>重新溶解干寡核苷酸探针混合在200µL 的 TE 缓冲器 (10 毫米三盐酸, 1 毫米 EDTA, pH 8.0) 创造一个探针库存25µM. 移上和下, 然后漩涡和离心机简要地混合好。</li>
		<li>为了避免冻结和解冻的股票溶液管, 使几个等分的股票解决方案, 对应的金额预计将使用在一个单一的实验运行。存储在-20 ° c 或以下的股票解决方案, 并保护免受光。 注: 探针组 5 nmol 可以提供多达250杂交反应。</li>
	</ol>
	</li>
	<li>杂交缓冲 (跟随剥皮器et al.7 ):
	<ol>
		<li>在50毫升聚丙烯锥底管上加入5毫升无核酸水。在水中加入1克葡聚糖硫酸酯, 并通过剧烈的涡流溶解。混合在一个轨道振动筛的内容, 直到气泡消失 (〜30分钟)。</li>
		<li>添加到解决方案3530µL 的去离子甲酰胺, 10 毫克的大肠杆菌tRNA, 1mL 的 20x SSC, 100 µL 200 毫米钒 ribonucleoside 复合体和40µL 50 毫克/毫升 BSA。通过加 DEPC 处理的水或无核酸的水, 将最终的体积带到10毫升, 然后漩涡溶液直到它是均匀的。 注意: 为了避免冻结和解冻的解决方案, 使几个等分的股票解决方案, 对应的金额预计将使用在一个单一的实验运行。储存在-20 ° c 的股票解决方案。在打开瓶子之前, 一定要让甲酰胺温暖到室温。为了减少背景, 建议校准最佳甲酰胺浓度 (10-40% v/v 比)。 
		警告:警告!甲酰胺是剧毒和已知的致。避免接触眼睛或皮肤, 吸入或吞食。穿着实验室大衣和防护手套, 在通风橱下处理。</li>
		<li>通过0.22 µm 过滤器对溶液进行灭菌。保持在等分和存储在-20 ° c, 长达一年。</li>
	</ol>
	</li>
	<li>在 1x pbs 中加入37% 甲醛 (v/v) 至 1x pbs (见 2.1.1), 在 1:8 v/v 比和涡旋上制备固定缓冲剂 (4.6% 甲醛)。 小心: 警告!甲醛是剧毒和已知的致癌物。穿着实验室大衣和防护手套, 在通风橱下处理。</li>
	<li>为 smFISH (2x ssc 中的20% 甲酰胺) 准备洗涤缓冲器, 将去离子甲酰胺加入到 2x ssc (见 2.1.2) 至1:4 伏/v 比和涡旋。</li>
	<li>通过添加5毫米半胱胺和氧清除剂 (7 µM 葡萄糖氧化酶、56 nM 过氧化氢酶和10% 葡萄糖 (w/v)) 清洗缓冲液 (2x SSC 中的20% 甲酰胺) 来制备 smFISH 的成像缓冲器</li>
	<li>缓冲风暴
	<ol>
		<li>准备缓冲 A, 加入50毫米的三盐酸和10毫米氯化钠, 以核酸无水。 注: 在 RT 中存储多年。</li>
		<li>准备缓冲 B, 加入50毫米的三盐酸盐, 10 毫米氯化钠, 和10% 核酸葡萄糖到无水。 注: 贮存时间为4° c, 可达一年。</li>
		<li>在1毫升的缓冲器 a 中溶解77毫克半胱胺, 以制备一个1米的溶液。 注: 可储存在4° c 1 月。</li>
		<li>将 8440 au (任意单位) 葡萄糖氧化酶和 70200 au 过氧化氢酶添加到1毫升缓冲 A, 准备 Gloxy 缓冲液。 注: 制作50x 库存解决方案, 可存放在4° c 1 月。</li>
		<li>通过在缓冲区 B 中添加 50 mM 缓冲区和 1x Gloxy 缓冲区, 准备风暴的成像缓冲器。 注: 风暴成像缓冲器由5毫米半胱胺, 氧清除剂 (7 µM 葡萄糖氧化酶和 56 nM 过氧化氢酶) 在50毫米三与10毫米氯化钠, 和10% 葡萄糖在 pH 8.0。在成像前准备, 它可以存储和使用在 RT 约2小时。</li>
	</ol>
	</li>
</ol>
3. 样品固定
<ol>
	<li>培养细菌细胞 (例如, e.</li></ol>大肠杆菌 MG1655) 在生长培养基的选择 (如, LB 中等) 过夜阿齐兹·伊南轨道振动筛在 260 rpm 和37° c。 注意: 要确定由于探针的非特异性结合而产生的背景, 应按照相同的步骤进行测量, 在这种情况下, 应在已删除感兴趣的基因的应变中进行测试 (请参见图 1和δsodB中的δgalk 图 2)。
	<li>在新鲜培养基中稀释隔夜培养 1:100, 测量其光学密度 (OD600) 在分光光度计中每〜 1 h, 其值为 0.2-0.4;4毫升的文化应该是足够的。 注: 如果所选显微镜没有相位对比度或差分干扰对比能力, 则大肠杆菌外膜可以用2μ m 脂溶性荧光标记 (见材料表) 标记, 应添加到细菌性悬浮20分钟前固定17。固定和进一步治疗是完成如下所述。必须注意选择具有不同发射谱的荧光标记, 以尽量减少与标记探针集的频谱重叠。</li>
	<li>颗粒细胞由离心5分钟在 4500 g 在4° c。从所有管子中取出上清液。</li>
	<li>加入1毫升的 1x PBS, 每管和重的颗粒, 以减少细胞聚集。然后轻轻地加入4毫升的固定缓冲液和涡旋。</li>
	<li>在 260 rpm 的轨道振动筛中孵育30分钟, 在24° c。</li>
	<li>颗粒细胞离心10分钟, 1000 克, 4 ° c。除去上清液。添加1毫升 1x PBS。</li>
	<li>将悬浮液转移到1.8 µL 锥底微离心管 (见材料表), 重复步骤3.6 次。</li>

4. 样品性
<ol>
	<li>非常小心地除去上清。如有必要, 使用小体积管, 除去颗粒外的所有液体。</li>
	<li>重在300µL DEPC 处理的水或无核酸水。添加350µL 高品位绝对乙醇 (99%), 并通过倒置管轻轻搅拌。再加入350µL 乙醇和混合, 以达到最终的乙醇浓度为 70% (v/v 比)。 小心: 警告!乙醇是易燃的。</li>
	<li>在室温 (RT) 或在4° c 一夜之间旋转 20 rpm 的试管回转试样。样品可贮存在4° c 至一周。</li>
</ol>
5. 杂交
<ol>
	<li>颗粒细胞离心7分钟, 750 x g, 4 ° c。除去上清液。</li>
	<li>在样品上加入1毫升20% 洗涤缓冲器, 并离开站立几分钟, 或吸管完全溶解颗粒。</li>
	<li>颗粒细胞离心7分钟, 在 750 x g, 4 ° c。</li>
	<li>用移轻轻地除去清液。如有必要, 使用小体积管除去颗粒外的所有液体。</li>
	<li>加入寡核苷酸探针集料溶液到杂交缓冲分, 使最终的寡核苷酸浓度为 250 nM;漩涡大力。 注意: 一个可以在同一单元格中同时标记两个或多个不同的目标基因。将两个目标的探测集添加到杂交缓冲中。必须注意选择具有不同发射光谱的染料, 以尽量减少频谱重叠 (例如、Cy3 和 Cy5)。确保用于风暴成像的样品与一个与合适的染料相结合的寡核苷酸探针集 (例如, 在材料表中) 杂交。</li>
	<li>在包含寡核苷酸探针的50µL 杂交缓冲中暂停样品 (见步骤 5.4), 同时避免产生气泡 (溶液相当粘性)。</li>
	<li>将样品隔夜放在30° c 的热块中。 注: 在此之后, 样品可在4° c 下贮存长达6月。</li>
</ol>
6. 洗涤
<ol>
	<li>使用一个新的离心管为每个样品的图像。添加1毫升洗涤缓冲器。</li>
	<li>添加10-15 µL 从杂交样品到新的管, 包含洗涤缓冲和混合/漩涡。</li>
	<li>颗粒细胞由离心7分钟, 750 x g 在4° c。除去上清液。 注意: 为减少背景, 孵育1小时在30° c。</li>
	<li>再洗两次 (重在1毫升洗涤缓冲液, 离心机和去除颗粒)。</li>
	<li>重在25µL 洗涤缓冲器。 注: 为了减少光漂白, 可以在洗涤缓冲液中添加一个氧清除系统 (2.6), 以提高分子荧光实验的染料稳定性6。</li>
</ol>
7. 成像样品的制备
注意: 细胞需要固定, 以便在荧光和相衬显微镜下进行适当的成像。以下方法可用于准备样品, 在不同的焦平面上可以对不同的视场进行成像。
<ol>
	<li>琼脂糖凝胶制剂
	<ol>
		<li>添加150毫克琼脂糖到10毫升 2x SSC 缓冲。不要将 2x SSC 添加到琼脂糖中, 否则它将无法正常溶解。</li>
		<li>每次10-15 秒的微波, 直到大气泡开始形成。放在一旁冷静几分钟。</li>
		<li>在幻灯片上放置一个分离的硅胶框架, 使幻灯片的中心保持曝光。</li>
		<li>在滑动中心放置一个10毫米宽 (1-2 毫米厚) 的刚性环, 并在其中沉积少量的凝胶, 用于封口。等待几分钟, 让它变硬。</li>
		<li>添加更多的凝胶, 直到形成一个高圆顶形状。等待10分钟, 使其硬化, 并删除环 (使用细镊子或任何其他方法)。</li>
		<li>沉积〜10μ l 洗涤的细菌细胞 (参见 6.5) 在胶凝体和封印与 #0 片幻灯片。</li>
	</ol>
	</li>
	<li>smFISH 室样品的制备
	<ol>
		<li>或者 (对步骤 7.1) 准备样品为成像通过固定洗涤细菌细胞 (参见 6.5) 在聚-d-赖氨酸被涂覆的玻璃底部一次性塑料培养皿。</li>
		<li>在黑暗中孵育样品, 在室温下, 在可视化之前过夜, 使细胞黏附在表面。在可视化清洗前用洗涤缓冲器 (. 5);用洗涤缓冲器 (2.5) 或 smFISH (2.6) 的成像缓冲器使样品湿润。</li>
	</ol>
	</li>
	<li>风暴室样品准备
	<ol>
		<li>准备在聚 d-赖氨酸涂层玻璃底部的成像风暴样品一次性塑料培养皿。</li>
		<li>在黑暗中, 在 RT, 一夜之间孵化样品。在形象化洗涤以前用洗涤的缓冲为 smFISH (2.5);并为风暴 (2.7.5) 添加成像缓冲器。</li>
	</ol>
	</li>
</ol>
8。字幕可视化
<ol>
	<li>具有一个机动阶段的广域荧光显微镜的图像细胞 (见材料表)。 注意: 对于风暴的修改, 使用标记有染料的细胞可以多循环的荧光发射。图像的样品使用3D 超分辨率成像系统 (例如, 请参阅材料表)。</li>
	<li>使用 (推荐) 60-100x; #62; 1.3 油浸相对比物镜与强光源, 如汞或金属卤化物灯;led 光源也推荐使用。</li>
	<li>使用标准的冷 CCD 相机, 理想的优化低光水平成像而不是速度 (我们使用16µm 像素大小的相机) (见材料表)。 注: 使用冷 EMCCD (电子倍增电荷耦合器件) 相机 CCD 相机是必要的, 以减少热噪声, 防止检测, 特别是单一分子。</li>
	<li>使用适合于所选荧光的筛选器集。</li>
	<li>为了确定适当的细胞边界, 在每个样品中获得不同视场的图像, 或者用相衬光学, 或者通过标记荧光外细胞膜。在不同的焦平面 (z 堆叠) 上获取图像 (通常13片被 250 nm 分开)。 注: 显微镜和过滤器的电控阶段应由商业 (见材料表) 或 in-house 软件控制, 可以按顺序捕获一堆图像。</li>
</ol>
9. 数据分析
<ol>
	<li>将图像堆栈转换为适当的数据分析格式。</li>
	<li>估计的目标 mRNA 的拷贝数从总强度的荧光病灶的细胞, 而不是从计数离散的 "斑点", 在其他现有的协议。</li>
	<li>使用开源软件18或 custom-built 程序进行图像分析。有关数据成像和分析的详细信息, 请参阅斯金纳et al.7</li>
</ol>

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作者没有什么可透露的。

我们在实验室测量了使用 smFISH 方法 2的大肠杆菌细胞中不同基因的转录数量。简而言之, 这个过程包括以下步骤: 细胞固定, 性的膜, 允许探针穿透, 探针杂交, 和样品成像使用标准的荧光显微镜。此过程基于以前发布的一些修改6,7,16。此前有报道称, smFISH 要求寡核苷酸探针的数量在48-72 范围内, 以达到在背景7以上的信号。我们已经表明, 这个数字可能实际上是较小的20, 这取决于感兴趣的基因序列、光学设置和特定的实验条件。
每个探针应该是17-22 核苷酸长, 以 inter-probe 分离至少二核苷酸和 GC 内容的 ~ 45%, 为了减少非特异性, 不相干捆绑的作用。某些探头可能无法绑定目标, 并且通过修改实验程序 (如固定条件和杂交21) 来优化绑定的整体效率。必须考虑的一个重要因素是荧光的选择。强烈建议用低易感性的染料来标记探针。光漂最小化可以通过优化曝光时间、光照源和图像采集的积分次数, 以及通过增加氧清除系统来实现。
非特异性结合事件的程度应通过进行 smFISH 实验来评估, 其中有靶基因被删除的菌株的细胞, 例如来自庆应的集合19。如果背景信号很高, 则应增加洗涤步骤的次数和持续时间, 或在30° c 为1小时的洗涤缓冲液中孵育细胞, 然后冲洗两次。此外, 为了减少背景, 建议在杂交和洗涤缓冲器中优化甲酰胺浓度 (10-40% v/v 比)。增加甲酰胺浓度减少非特异性结合, 但也可能减少探针对靶 mRNA 的结合。在杂交过程中, 完全清除上清液是很重要的;稀杂交缓冲可能导致标记效率降低。此外, 目标 rna 必须足够长, 以获得一个 well-localized 点以上的背景。最近通过探头的主干修改来改善信噪比和结合特异性, 现在可以检测更短的 RNA 片段, 如真核 rna 或细菌小编码 rna2,10. 我们建议用定量 PCR 方法验证 smFISH 获得的平均拷贝数716。
我们在这份手稿中已经表明, 这种方法可以修改, 以小的变化, 以研究的转录在大肠杆菌的本地化与更高的光学分辨率使用随机光学重建显微镜 (风暴)11。
总之, smFISH 是一种通用的 RNA 光照方法, 它允许直接测量转录数的细胞变异和在真核细胞和原的细胞中的目标转录的定位。它提供了有关基因表达的基本过程的定量信息, 可以很容易地实现风暴成像。

随机性是基因表达的一个基本的和不可避免的方面, 并引起细胞细胞的异质性1, 无论是在转录和蛋白质水平上的2,3。在定义良好的条件下量化细胞间的变异性, 为基因表达及其调控的基本过程提供了一个独特的窗口。一个重要来源的细胞细胞异质性细菌发生在转录水平。成绩单的数量不仅因转录的随机性而异, 也因后的过程, 如由小 rna 和 RNAases2的调节。一种直接以定量方式访问这种异质性的方法是通过荧光标记特定基因在 smFISH 中的单个转录。这种方法允许检测和特定的 RNA 分子的亚细胞定位固定的, 个别的细菌单元4。基因与一组荧光标记的 ~ 20 基长的寡核苷酸杂交, 目的是选择性地绑定到感兴趣的记录5,6。多重标记可确保在背景荧光之上检测, 而单个 mRNA 分子在荧光显微镜下显示为衍射限制斑点7 (见图 1)。还有其他方法来标记 mRNA 分子, 其中互补齐聚物探针携带共轭半 (如, 生物素或辛), 被检测使用第二荧光标记的记者技术8。
除了 smFISH, 还有其他方法可以提供关于成绩单的定量信息。一些, 如北印迹或定量 PCR, 探针的体积, 从而可以测量既不是 mRNA 的数量, 也不是他们的位置在单个细胞。因此, 这些方法不适合量化细胞的变异性。最近的一种基于图像的技术, 允许定量的 rna 的副本数量和细胞内的位置, 称为复用误差鲁棒荧光原位杂交 (MERFISH) 已经发展。MERFISH 是基于一个独特的条形码的分配, 由固定数量的荧光标记的寡核苷酸探针的定义组合。这些条形码在 smFISH 测量的连续的圆读出来, 与漂白跟随每轮杂交, 从而增加吞吐量由二个数量级9,10。这种技术需要一个自动流体处理系统和适当的探头设置设计。
多重荧光标记的个人成绩单组合, 连同新的超分辨率技术, 如随机光学重建显微镜 (风暴)11, 使分辨率增加十倍转录的亚细胞定位。在风暴中, 一个合适的组合荧光探针和成像缓冲允许多个周期的荧光发射每个探针分子 (闪烁)。风暴也可用于图像的大肠杆菌转录和观察 RNA 的全基因组的空间组织, 通过标签同时所有的记录的兴趣,12。
上述所有的单细胞方法都是基于固定细胞的成像记录。因此, 他们没有提供任何关于细胞内转录物的动力学特性的信息。要跟踪活细胞的转录13, 基因可以标记的基因的融合的利益, 以一个数组的绑定网站。后者由 RNA 结合的蛋白质认可, 例如噬菌体 MS2 外套蛋白质, 被熔化对荧光蛋白质例如绿色萤光蛋白质 (GFP)10,14,15。
在这里, 我们描述一种方法, 标记个别基因与一组荧光标记的 DNA 探针, 用于 smFISH 实验, 特别是在大肠杆菌中使用。此外, 我们表明, 相同的标记方案可以用于风暴测量, 小修改。

<table><tbody><tr><td>Dextran sulfate sodium salt</td><td>Sigma-Aldrich</td><td>D8906</td><td></td></tr><tr><td>Pure Ethanol, 99.5%, ACS reagent, absolute</td><td>Mallinckrodt Baker - Avantor</td><td>8025.25</td><td></td></tr><tr><td>Diethylpyrocarbonate (DEPC)</td><td>Sigma-Aldrich</td><td>D5758</td><td></td></tr><tr><td>RNase-free 20X SSC</td><td>Life Technologies/Ambion</td><td>AM9763</td><td></td></tr><tr><td>RNase-free 10X PBS</td><td>Life Technologies/Ambion</td><td>AM9625</td><td></td></tr><tr><td>TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) BioUltra, for molecular biology</td><td>Sigma-Aldrich</td><td>93283</td><td></td></tr><tr><td>nuclease-free water</td><td>Thermo Fisher Scientific</td><td>10977035</td><td></td></tr><tr><td>Formaldehyde solution for molecular biology, 36.5-38% in water</td><td>Sigma-Aldrich</td><td>F8775</td><td></td></tr><tr><td>Deionized formamide, nuclease free</td><td>Thermo Fisher Scientific/Ambion</td><td>AM9342</td><td></td></tr><tr><td>E. coli tRNA (ribonucleic acid, transfer type xx from escherichia)</td><td>Sigma-Aldrich</td><td>R1753-500UN</td><td></td></tr><tr><td>UltraPure BSA (50mg/ml)</td><td>Thermo Fisher Scientific/Ambion</td><td>AM2616</td><td></td></tr><tr><td>Vanadyl-ribonucleoside complex,VRC, 200 mM</td><td>New England Biolab</td><td>S1402S</td><td></td></tr><tr><td>Poly-L-Lysine</td><td>Sigma</td><td>P4707</td><td></td></tr><tr><td>Cysteamine and oxygen</td><td>Sigma-Aldrich</td><td>30070</td><td></td></tr><tr><td>Glucose Oxidase from Aspergillus niger, Type VII, 50KU</td><td>Sigma-Aldrich</td><td>G2133</td><td></td></tr><tr><td>Catalase</td><td>Sigma-Aldrich</td><td>C40</td><td></td></tr><tr><td>D-glucose</td><td>Sigma-Aldrich</td><td>G8270</td><td></td></tr><tr><td>D-fucose</td><td>Sigma-Aldrich</td><td>F8150</td><td></td></tr><tr><td>Vybrant DiO Cell-Labeling Solution</td><td>Life Technologies</td><td>V2286</td><td></td></tr><tr><td>Agarose,low melting reagent</td><td>Sigma-Aldrich</td><td>A9414</td><td></td></tr><tr><td>Adhesive silicone isolator 24-2mm Dia. X 1.8 mm depth JTR24R-A2-2.0</td><td>Grace Bio-Labs</td><td>JTR24R-A2-2.0&lt;span&gt; &lt;/span&gt;666208</td><td></td></tr><tr><td>poly-D-lysine-coated glass bottom Glass Bottom Culture Dishes</td><td>MatTek Corporation</td><td>P35GC-1.5-14-C</td><td></td></tr><tr><td>Super life nitrile powder free examination gloves</td><td>Supermax</td><td>TC-N-9889</td><td></td></tr><tr><td>Brand sterilization incubator tape</td><td>Sigma-Aldrich</td><td>BR61750</td><td></td></tr><tr><td>Microcentrifuge tubes (1.8 ml)</td><td>Axygen - Corning Life Sciences</td><td>MCT-175C</td><td></td></tr><tr><td>Falcon round-bottom polypropylene tubes (14 ml)</td><td>BD Biosciences</td><td>352059</td><td></td></tr><tr><td>Conical-bottom centrifuge polypropylene tubes (50 ml)</td><td>Corning</td><td>430828</td><td></td></tr><tr><td>Serological pipettes (Corning 5 ml)</td><td>Corning Life Sciences</td><td>4051</td><td></td></tr><tr><td>Serological pipettes (Corning 10 ml)</td><td>Corning Life Sciences</td><td>4488</td><td></td></tr><tr><td>Serological pipettes (Corning 25 ml)</td><td>Corning Life Sciences</td><td>4251</td><td></td></tr><tr><td>Spectrophotometer cuvettes</td><td>Sarstedt</td><td>67.742</td><td></td></tr><tr><td>RNase-free pipette tips 0.2 - 20 &amp;mu;l</td><td>FroggaBio</td><td>FT20</td><td></td></tr><tr><td>RNase-free pipette tips 10 - 200 &amp;mu;l</td><td>Axigene/corning</td><td>TF-200</td><td></td></tr><tr><td>RNase-free pipette tips 100 - 1000 &amp;mu;l</td><td>FroggaBio</td><td>FT1000</td><td></td></tr><tr><td>RNase-free pipette tips 100 - 1000 &amp;mu;l</td><td>Sorenson</td><td>14200</td><td></td></tr><tr><td>Syringe disposile 10 mL needle G-21</td><td>Becton Dickinson, Biosciences</td><td>BD-309643</td><td></td></tr><tr><td>Minisart 0.2 um Syringe Filter</td><td>Sartorius</td><td>16534 K</td><td></td></tr><tr><td>Nikon instruments microscope type A immersion oil A, 8cc</td><td>Nikon</td><td>MXA20233</td><td></td></tr><tr><td>Microscope slides 76 x26, 3&quot;x1&quot;x1mm</td><td>Thermo Fisher Scientific</td><td>421-004ET</td><td></td></tr><tr><td>#0 coverslip slide 24x60</td><td>Thermo Fisher Scientific/Menzel</td><td>BNBB024060A0</td><td></td></tr><tr><td>Orbital shaker</td><td>M.R.C</td><td>TOU-50</td><td></td></tr><tr><td>Hot block</td><td>M.R.C</td><td></td><td></td></tr><tr><td>Vortex</td><td>Fried Electric Company</td><td>G-560-E</td><td></td></tr><tr><td>Microcentrifuge</td><td>Eppendorf</td><td>5427R</td><td></td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td>5810R</td><td></td></tr><tr><td>Portable Pipet-Aid XP2, Pipette Controller</td><td>Drummond Scientific Company</td><td>4-000-501-I</td><td></td></tr><tr><td>OD600 Spectrophotometer for Bacterial Growth Rates DiluPhotometer</td><td>Midsci</td><td>OD600-10</td><td></td></tr><tr><td>iXon X3 EMCCD camera</td><td>Andor</td><td>DU-897E-CS0-#BV</td><td> </td></tr><tr><td>Eclipse Ti microscope</td><td>Nikon</td><td>MEA53100</td><td> </td></tr><tr><td>CFI plan apochromat DM 100X oil objective lambda PH-3 N.A 1.45 WD 0.13</td><td>Nikon</td><td>MRD31905</td><td> </td></tr><tr><td>Filter set (TRITC/CY3): EX - ET545/30X; EM - ET620/60M; BS - T570LP</td><td>Nikon</td><td>49005</td><td> </td></tr><tr><td>Filter set (CY5): EX - ET640/30X; EM - ET690/50M; BS - T660P</td><td>Nikon</td><td>49009</td><td> </td></tr><tr><td>Nis-elements AR auto reaserch software</td><td>Nikon</td><td>MQS31000</td><td> </td></tr><tr><td>STORM microscope</td><td>Vutara</td><td>SR-200</td><td> </td></tr><tr><td>NA 1.2 water immersion objective&lt;span&gt; &lt;/span&gt;</td><td>Olympus</td><td> </td><td> </td></tr><tr><td>SRX image acquisition and analysis software</td><td>Vutara</td><td></td><td></td></tr><tr><td>Evolve 512 EMCCD camera</td><td>Photometrics</td><td></td><td></td></tr><tr><td>Stellaris&amp;reg; FISH Probes, Custom Assay with CAL Fluor&amp;reg; Red 590 Dye</td><td>Biosearch Technologies Inc</td><td>SMF-1083-5</td><td></td></tr><tr><td>&lt;strong&gt;Probe Sequence for galK mRNA:&lt;/strong&gt;</td><td> </td><td> </td><td></td></tr><tr><td>gtgttttttctttcagactc</td><td> </td><td> </td><td></td></tr><tr><td>tagccaaatgcgttggcaaa</td><td> </td><td> </td><td></td></tr><tr><td>ctgaatggtgtgagtggcag</td><td> </td><td> </td><td></td></tr><tr><td>caccaatcaaattcacgcgg</td><td> </td><td> </td><td></td></tr><tr><td>acgaaaccgtcgttgtagtc</td><td> </td><td> </td><td></td></tr><tr><td>tgataatcaatcgcgcaggg</td><td> </td><td> </td><td></td></tr><tr><td>gtggtgcacaactgatcacg</td><td> </td><td> </td><td></td></tr><tr><td>acgcgaactttacggtcatc</td><td> </td><td> </td><td></td></tr><tr><td>gctgattttcataatcggct</td><td> </td><td> </td><td></td></tr><tr><td>gcatcgagggaaaactcgtc</td><td> </td><td> </td><td></td></tr><tr><td>gttttcatgtgcgacaatgg</td><td> </td><td> </td><td></td></tr><tr><td>cacgccacgaacgtagttag</td><td> </td><td> </td><td></td></tr><tr><td>ttacgcagttgcagatgttt</td><td> </td><td> </td><td></td></tr><tr><td>tccagtgaagcggaagaact</td><td> </td><td> </td><td></td></tr><tr><td>tgctgcaatacggttccgac</td><td> </td><td> </td><td></td></tr><tr><td>gtccagcggcagatgataaa</td><td> </td><td> </td><td></td></tr><tr><td>tgaccgttaagcgcgatttg</td><td> </td><td> </td><td></td></tr><tr><td>agcctacaaactggttttct</td><td> </td><td> </td><td></td></tr><tr><td>gcggaaattagctgatccat</td><td> </td><td> </td><td></td></tr><tr><td>aaggcatgatctttcttgcc</td><td> </td><td> </td><td></td></tr><tr><td>cagtgagcggcaatcgatca</td><td> </td><td> </td><td></td></tr><tr><td>tgggcatggaaactgctttg</td><td> </td><td> </td><td></td></tr><tr><td>gatgatgacgacagccacac</td><td> </td><td> </td><td></td></tr><tr><td>gggtacgtttgaagttactg</td><td> </td><td> </td><td></td></tr><tr><td>gtgttgtattcgctgccaac</td><td> </td><td> </td><td></td></tr><tr><td>ggtttcgcactgttcacgac</td><td> </td><td> </td><td></td></tr><tr><td>tggctgctggaagaaacgcg</td><td> </td><td> </td><td></td></tr><tr><td>ttcaatggtgacatcacgca</td><td> </td><td> </td><td></td></tr><tr><td>catgcgcaacagcgttgaac</td><td> </td><td> </td><td></td></tr><tr><td>ggcgttttcagtcagtatat</td><td> </td><td> </td><td></td></tr><tr><td>atacgtttcaggtcgccttg</td><td> </td><td> </td><td></td></tr><tr><td>tgagactccgccatcaactc</td><td> </td><td> </td><td></td></tr><tr><td>gaaatcatcgcgcatagagg</td><td> </td><td> </td><td></td></tr><tr><td>caatttgcggcacggtgatt</td><td> </td><td> </td><td></td></tr><tr><td>ttgacgatttctaccagagt</td><td> </td><td> </td><td></td></tr><tr><td>acctttgtcgccaatcacag</td><td> </td><td> </td><td></td></tr><tr><td>ggatcagcgcgacgatacag</td><td> </td><td> </td><td></td></tr><tr><td>atattgttcagcgacagctt</td><td> </td><td> </td><td></td></tr><tr><td>gtctctttaatacctgtttt</td><td> </td><td> </td><td></td></tr><tr><td>ctccttgtgatggtttacaa</td><td> </td><td> </td><td></td></tr><tr><td>Stellaris&amp;reg; FISH Probes, Custom Assay with Quaser Fluor&amp;reg; Red 670 Dye</td><td>Biosearch Technologies Inc</td><td>SMF-1083-5</td><td></td></tr><tr><td>&lt;strong&gt;Probe Sequence for sodB mRNA:&lt;/strong&gt;</td><td> </td><td> </td><td></td></tr><tr><td>gtgttttttctttcagactc</td><td> </td><td> </td><td></td></tr><tr><td>tagccaaatgcgttggcaaa</td><td> </td><td> </td><td></td></tr><tr><td>ctgaatggtgtgagtggcag</td><td> </td><td> </td><td></td></tr><tr><td>caccaatcaaattcacgcgg</td><td> </td><td> </td><td></td></tr><tr><td>acgaaaccgtcgttgtagtc</td><td> </td><td> </td><td></td></tr><tr><td>tgataatcaatcgcgcaggg</td><td> </td><td> </td><td></td></tr><tr><td>gtggtgcacaactgatcacg</td><td> </td><td> </td><td></td></tr><tr><td>acgcgaactttacggtcatc</td><td> </td><td> </td><td></td></tr><tr><td>gctgattttcataatcggct</td><td> </td><td> </td><td></td></tr><tr><td>gcatcgagggaaaactcgtc</td><td> </td><td> </td><td></td></tr><tr><td>gttttcatgtgcgacaatgg</td><td> </td><td> </td><td></td></tr><tr><td>cacgccacgaacgtagttag</td><td> </td><td> </td><td></td></tr><tr><td>ttacgcagttgcagatgttt</td><td> </td><td> </td><td></td></tr><tr><td>tccagtgaagcggaagaact</td><td> </td><td> </td><td></td></tr><tr><td>tgctgcaatacggttccgac</td><td> </td><td> </td><td></td></tr><tr><td>gtccagcggcagatgataaa</td><td> </td><td> </td><td></td></tr><tr><td>tgaccgttaagcgcgatttg</td><td> </td><td> </td><td></td></tr><tr><td>agcctacaaactggttttct</td><td> </td><td> </td><td></td></tr><tr><td>gcggaaattagctgatccat</td><td> </td><td> </td><td></td></tr><tr><td>aaggcatgatctttcttgcc</td><td> </td><td> </td><td></td></tr><tr><td>cagtgagcggcaatcgatca</td><td> </td><td> </td><td></td></tr><tr><td>tgggcatggaaactgctttg</td><td> </td><td> </td><td></td></tr><tr><td>gatgatgacgacagccacac</td><td> </td><td> </td><td></td></tr><tr><td>gggtacgtttgaagttactg</td><td> </td><td> </td><td></td></tr><tr><td>gtgttgtattcgctgccaac</td><td> </td><td> </td><td></td></tr><tr><td>ggtttcgcactgttcacgac</td><td> </td><td> </td><td></td></tr><tr><td>tggctgctggaagaaacgcg</td><td> </td><td> </td><td></td></tr><tr><td>ttcaatggtgacatcacgca</td><td> </td><td> </td><td></td></tr><tr><td>catgcgcaacagcgttgaac</td><td> </td><td> </td><td></td></tr><tr><td>ggcgttttcagtcagtatat</td><td> </td><td> </td><td></td></tr><tr><td>atacgtttcaggtcgccttg</td><td> </td><td> </td><td></td></tr><tr><td>tgagactccgccatcaactc</td><td> </td><td> </td><td></td></tr><tr><td>gaaatcatcgcgcatagagg</td><td> </td><td> </td><td></td></tr><tr><td>caatttgcggcacggtgatt</td><td> </td><td> </td><td></td></tr><tr><td>ttgacgatttctaccagagt</td><td> </td><td> </td><td></td></tr><tr><td>acctttgtcgccaatcacag</td><td> </td><td> </td><td></td></tr><tr><td>ggatcagcgcgacgatacag</td><td> </td><td> </td><td></td></tr><tr><td>atattgttcagcgacagctt</td><td> </td><td> </td><td></td></tr><tr><td>gtctctttaatacctgtttt</td><td> </td><td> </td><td></td></tr><tr><td>ctccttgtgatggtttacaa</td><td> </td><td> </td><td></td></tr></tbody></table>

method for labeling transcripts in individual escherichia coli cells for single-molecule fluorescence in situ hybridization experiments

一种方法被描述为标记单独信使 RNA (mRNA) 抄本在固定的细菌为使用在分子荧光原位杂交 (smFISH) 实验在大肠杆菌. smFISH 允许测量感兴趣基因的 mRNA 拷贝数的细胞变异, 以及转录的亚细胞位置。所涉及的主要步骤是固定的细菌细胞培养, 性的细胞膜, 和杂交的目标抄本与一套商业可用的短荧光标记寡核苷酸探针。smFISH 可以允许对同一细胞中的多个基因的转录进行成像, 并在不同荧光标记之间的频谱重叠施加限制。在下面所示的协议完成后, 细胞可以很容易地成像使用显微镜和适合低强度的荧光相机。这些图像, 连同从相衬框架的分割获得的细胞轮廓, 或从细胞膜染色, 允许计算的 mRNA 拷贝数分布的样本细胞使用开源或定制的软件。本文所描述的标记方法也可以应用于随机光学重建显微镜 (风暴) 的图像记录。

我们进行了 smFISH 测量的 galK 和 sodB 转录在大肠杆菌细胞。抄本与一组与目标序列互补的特定序列杂交, 每个探针被共轭到一个单一的荧光分子 (见材料表)。MG1655 野生型大肠杆菌菌株的荧光和相衬图像 (或 JW0740)19 galK-已删除的应变 (ΔgalK) 被暴露到2毫米 D-藻显示在图 1A和1B中。在接触 d-藻后, 细胞被固定在至少3小时。图 1A (top) 中的面板显示出13的帧, 对应于单元格轮廓在焦点上的高度, 表示galK感应对可变胞外 d-藻浓度的响应程度。荧光图像中细胞内的斑点对应于单个或少量的 galk 转录, 并通过量化局部荧光来测定每个细胞中的转录数。在大多数细胞中都有许多斑点, 当2毫米 d-藻被添加到细菌培养中, 而在缺乏胞外 d-藻的情况下会发现一些斑点。在ΔgalK应变中, 没有按预期在背景上观察到斑点。表面标记与亲油性荧光标记显示在图 1B (底部)。
此外, 我们还在大肠杆菌中拍摄了在对数生长阶段的 LB 培养基中生长的细菌培养的sodB记录. smFISH 的荧光和相衬图像在 JW1648 (京王集合) 中的表现19 sodB 删除的应变 (ΔsodB) 显示在图 2A中。图 2A (top) 中的面板显示了一个13帧, 对应于单元格轮廓在焦点中的高度, 表示sodB的级别。在ΔsodB应变中, 没有按预期在背景上观察到斑点。
同样的文化是由风暴 (顶部) 成像, 并通过表面标记的细胞的周长确定了使用亲油性荧光标记, 以说明sodB在细胞内的本地化。染色的膜允许准确定位的转录在细胞 (图 2B)。ΔsodB应变作为背景信号, 并确定最小簇大小 (请参见图 2B)。smFISH2和风暴的平均成绩单数是可比较的。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56600/56600fig1.jpg"> 
图 1: D-藻对使用 smFISH 进行可视化的 galK 记录的影响.(A)顶部: 在单个大肠杆菌单元格中 smFISH galK mRNA 的荧光图像. 用或不带2毫米 d-藻生长了 MG1655 的野生型应变或 JW0740 (京王集合) galK-删除应变 (ΔgalK)19 。galK 抄本被杂交了到互补探针共轭与染料光学上等效 Cy3 (参见材料表)。底部: 相衬的相同的视场。(B)在固定前20分钟, 用或不带2毫米 d-藻的细胞生长并标记为2μ m 的亲油性膜染料 (见材料表)。上图: 用 smFISH galK mRNA 的荧光图像。图像是由一个商业软件控制的显微镜使用 100× N. 1.45 油浸相对比物镜 (见材料表) 和 EMCCD 相机 (见材料表)。所用的过滤器如材料表所述。在每个切片的 2 s 积分时间后, 获得了一个13切片和 250 nm 的荧光图像间距的 z 栈。中间: 与相衬成像相同的视野。底部: 带有亲油性荧光标记的细胞, 在材料表中描述适当的过滤器。<a href="//ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56600/56600fig1large.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/56600/56600fig2.jpg"> 
图 2: 在 smFISH 和暴风中使用同一探头设置的 sodB 记录的成像.(A)顶部: 在单个大肠杆菌单元格中 smFISH sodB mRNA 的荧光图像. MG1655 野生型应变 (小波) 或 JW1648 (京王收集) sodB-删除应变 (ΔsodB)19在 LB 介质中生长。sodB记录被杂交到互补探针, 与染料光学等同的 Cy5 (见材料表)。底部: 相衬的相同的视场。(B)在同一视场中由风暴 (670 nm 发射) 和细胞膜快照的荧光分子 (单个有色点) 的叠加。该细胞被标记为亲油性染料, 激发与氩激光在 488 nm 1% 的最大功率 (0.05 kW/cm2) 与发射峰值在 510 nm 和影像与适当的过滤器 (见材料表)。重量和sodB细胞的生长, 如上所述, 并标记为2μ m 亲油性膜染料 (见材料表) 20 分钟前固定。超分辨率图像用商业显微镜记录 (见材料表)。标签与染料光学等效的 Cy5 被本地化使用的激光在 647 nm 的成像缓冲区风暴。图像记录使用 60x, NA 1.2 水浸泡目标 (见材料表) 和 EMCCD 相机 (见材料表) 与增益设置在 50, 帧率在50赫兹, 最大功率647和 405 nm 激光器设置在5和 0.05 kW/cm2分别.获得的帧总数为8000。使用商业软件分析数据 (见材料表)。<a href="//ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56600/56600fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

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Method for Labeling Transcripts in Individual Escherichia coli Cells for Single-molecule Fluorescence In Situ Hybridization Experiments

单分子荧光原位杂交实验中单个大肠埃希菌细胞的转录标记方法

这篇手稿描述了一种方法, 标记个别信使 RNA (mRNA) 转录与荧光标记的 DNA 探针, 用于分子荧光原位杂交 (smFISH) 实验在大肠杆菌. smFISH 是一种可视化方法, 它允许在固定的单个细胞中同时检测、定位和量化单个 mRNA 分子。

A method is described for labeling individual messenger RNA (mRNA) transcripts in fixed bacteria for use in single-molecule fluorescence in situ ...

method-for-labeling-transcripts-individual-escherichia-coli-cells-for

Universidad San Sebastian

Research

JoVE Journal

Biology

Method for Labeling Transcripts in Individual Escherichia coli Cells for Single-molecule Fluorescence In Situ Hybridization Experiments

8.2K 次观看.  Weizmann Institute of Science. 这篇手稿描述了一种方法, 标记个别信使 RNA (mRNA) 转录与荧光标记的 DNA 探针, 用于分子荧光原位杂交 (smFISH) 实验在大肠杆菌. smFISH 是一种可视化方法, 它允许在固定的单个细胞中同时检测、定位和量化单个 mRNA 分子。

视频: Method for Labeling Transcripts in Individual Escherichia coli Cells for Single-molecule Fluorescence In Situ Hybridization Experiments

Single Molecule Fluorescence In Situ Hybridization (smFISH) Analysis in Budding Yeast Vegetative Growth and Meiosis

Single molecule fluorescence in situ hybridization (smFISH) is a powerful technique to study gene expression in single cells due to its ability to detect and count individual RNA molecules. Complementary to deep sequencing-based methods, smFISH provides information about the cell-to-cell variation in transcript abundance and the subcellular localization of a given RNA. Recently, we have used smFISH to study the expression of the gene&#160;NDC80&#160;during meiosis in budding yeast, in which two transcript isoforms exist and the short transcript isoform has its entire sequence shared with the long isoform. To confidently identify each transcript isoform, we optimized known smFISH protocols and obtained high consistency and quality of smFISH data for the samples acquired during budding yeast meiosis. Here, we describe this optimized protocol, the criteria that we use to determine whether high quality of smFISH data is obtained, and some tips for implementing this protocol in&#160;other yeast strains and growth conditions.

Single Molecule Fluorescence In Situ Hybridization smFISH Analysis in Budding Yeast Vegetative Growth and Meiosis

This single molecule fluorescence in situ hybridization protocol is optimized to quantify the number of RNA molecules in budding yeast during vegetative growth and meiosis.

单分子荧光原位杂交 (smFISH) 在发芽酵母营养生长和减数分裂中的分析

Single molecule fluorescence in situ hybridization (smFISH) is a powerful technique to study gene expression in single cells due to its ability to detect ...

科研

遗传学

Use of Single Molecule Fluorescent In Situ Hybridization (SM-FISH) to Quantify and Localize mRNAs in Murine Oocytes

Current methods routinely used to quantify mRNA in oocytes and embryos include digital reverse-transcription polymerase chain reaction (dPCR), quantitative, real-time RT-PCR (RT-qPCR) and RNA sequencing. When these techniques are performed using a single oocyte or embryo, low-copy mRNAs are not reliably detected. To overcome this problem, oocytes or embryos can be pooled together for analysis; however, this often leads to high variability amongst samples. In this protocol, we describe the use of fluorescence in situ hybridization (FISH) using branched DNA chemistry. This technique identifies the spatial pattern of mRNAs in individual cells. When the technique is coupled with Spot Finding and Tracking&#160;computer software, the abundance of mRNAs in the cell can also be quantified. Using this technique, there is reduced variability within an experimental group and fewer oocytes and embryos are required to detect significant differences between experimental groups. Commercially available branched-DNA SM-FISH kits have been optimized to detect mRNAs in sectioned tissues or adherent cells on slides. However, oocytes do not effectively adhere to slides and some reagents in the kit were too harsh resulting in oocyte lysis. To prevent this lysis, several modifications were made to the FISH kit. Specifically, oocyte permeabilization and wash&#160;buffers designed for the immunofluorescence of oocytes and embryos replaced the proprietary buffers. The permeabilization, washes, and incubations with probes and amplifier were performed in 6-well plates and oocytes were placed on slides at the end of the protocol using mounting media. These modifications were able to overcome the limitations of the commercially available kit, in particular, the oocyte lysis. To accurately and reproducibly count the number of mRNAs in individual oocytes, computer software was used. Together, this protocol represents an alternative to PCR and sequencing to compare the expression of specific transcripts in single cells.

Use of Single Molecule Fluorescent In Situ Hybridization SM-FISH to Quantify and Localize mRNAs in Murine Oocytes

To reproducibly count the numbers of mRNAs in individual oocytes, single molecule RNA fluorescence in situ hybridization (RNA-FISH) was optimized for non-adherent cells. Oocytes were collected, hybridized with the transcript specific probes, and quantified using an image quantification software.

单分子荧光原位杂交 (SM-FISH) 在小鼠卵母细胞中 Mrna 的定量和定位

Current methods routinely used to quantify mRNA in oocytes and embryos include digital reverse-transcription polymerase chain reaction (dPCR), ...

Site-Directed Mutagenesis for In Vitro and In Vivo Experiments Exemplified with RNA Interactions in Escherichia Coli

Site-directed mutagenesis is a technique used to introduce specific mutations in DNA to investigate the interaction between small non-coding ribonucleic acid (sRNA) molecules and target messenger RNAs (mRNAs). In addition, site-directed mutagenesis is used to map specific protein binding sites to RNA. A 2-step and 3-step PCR based introduction of mutations is described. The approach is relevant to all protein-RNA and RNA-RNA interaction studies. In short, the technique relies on designing primers with the desired mutation(s), and through 2 or 3 steps of PCR synthesizing a PCR product with the mutation. The PCR product is then used for cloning. Here, we describe how to perform site-directed mutagenesis with both the 2- and 3-step approach to introduce mutations to the sRNA, McaS, and the mRNA, csgD, to investigate RNA-RNA and RNA-protein interactions. We apply this technique to investigate RNA interactions; however, the technique is applicable to all mutagenesis studies (e.g., DNA-protein interactions, amino-acid substitution/deletion/addition). It is possible to introduce any kind of mutation except for non-natural bases but the technique is only applicable if a PCR product can be used for downstream application (e.g., cloning and template for further PCR).

Site-Directed Mutagenesis for In Vitro and In Vivo Experiments Exemplified with RNA Interactions in Escherichia Coli

Site-directed mutagenesis is a technique used to introduce specific mutations in deoxyribonucleic acid (DNA). This protocol describes how to do site-directed mutagenesis with a 2-step and 3-step polymerase chain reaction (PCR) based approach, which is applicable to any DNA fragment of interest.

体外和体内实验的位定向诱变-以大肠杆菌rna相互作用为例

Site-directed mutagenesis is a technique used to introduce specific mutations in DNA to investigate the interaction between small non-coding ribonucleic ...

生物化学

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 ...

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 ...

<meta charset="utf8"></head><body>用于 smFRET-TIRF 研究的 RNA 双色末端标记和囊泡封装

Fluorescent End-Labeling and Encapsulation of Long RNAs for Single-Molecule FRET-TIRF Microscopy

Single-molecule F&#246;rster Resonance Energy Transfer (smFRET) excels in studying dynamic biomolecules by allowing precise observation of their conformational changes over time. To monitor RNA dynamics with smFRET, we developed a method to covalently label RNAs at their termini with a FRET pair of fluorophores. This direct end-labeling strategy targets the 5&#39;-phosphate by carbodiimide (EDC)/N-hydroxysuccinimide (NHS) activation and the 3&#39;-ribose by periodate oxidation, which can be adapted to other RNAs regardless of their size and sequence to study them independently of artificial modifications. Furthermore, the 5&#39;-EDC/NHS activation is of general interest to all nucleic acids with a 5&#39;-phosphate. The use of commercially available chemicals eliminates the need to synthesize RNA-specific probes.
Total Internal Reflection Fluorescence (TIRF) microscopy requires the surface-immobilized molecules of interest to be within the evanescent field to be illuminated. A sophisticated way of keeping the RNA molecules within the evanescent field is to encapsulate them in phospholipid vesicles. Encapsulation benefits from the best of both worlds, tethering the molecule to the surface while enabling free diffusion of the molecule. We ensure that each vesicle contains only a single RNA molecule, enabling single-molecule imaging. Upon dual-end labeling and encapsulation of the RNA of interest, smFRET measurements offer a dynamic and detailed view of RNA behavior.

<meta charset="utf8"></head><body>用于单分子 FRET-TIRF 显微镜的长 RNA 的荧光末端标记和封装

This article describes the dual-color labeling of long RNAs at termini positions and their surface immobilization via encapsulation in phospholipid vesicles for single-molecule FRET TIRF microscopy applications. Combining these techniques enables precise visualization and analysis of RNA dynamics at the single-molecule level.

用于单分子 FRET-TIRF 显微镜的长 RNA 的荧光末端标记和封装

Single-molecule F&#246;rster Resonance Energy Transfer (smFRET) excels in studying dynamic biomolecules by allowing precise observation of their ...

Analysis of Single-cell Gene Transcription by RNA Fluorescent In Situ Hybridization (FISH)

Adhesion of Plasmodium falciparum infected erythrocytes (IE) to human endothelial receptors during malaria infections is mediated by expression of PfEMP1 protein variants encoded by the var genes.
The haploid P. falciparum genome harbors approximately 60 different var genes of which only one has been believed to be transcribed per cell at a time during the blood stage of the infection. How such mutually exclusive regulation of var gene transcription is achieved is unclear, as is the identification of individual var genes or sub-groups of var genes associated with different receptors and the consequence of differential binding on the clinical outcome of P. falciparum infections. Recently, the mutually exclusive transcription paradigm has been called into doubt by transcription assays based on individual P. falciparum transcript identification in single infected erythrocytic cells using RNA fluorescent in situ hybridization (FISH) analysis of var gene transcription by the parasite in individual nuclei of P. falciparum IE1.
Here, we present a detailed protocol for carrying out the RNA-FISH methodology for analysis of var gene transcription in single-nuclei of P. falciparum infected human erythrocytes. The method is based on the use of digoxigenin- and biotin- labeled antisense RNA probes using the TSA Plus Fluorescence Palette System2 (Perkin Elmer), microscopic analyses and freshly selected P. falciparum IE. The in situ hybridization method can be used to monitor transcription and regulation of a variety of genes expressed during the different stages of the P. falciparum life cycle and is adaptable to other malaria parasite species and other organisms and cell types.

Analysis of Single-cell Gene Transcription by RNA Fluorescent In Situ Hybridization FISH

Fluorescent in situ hybridization (FISH) to identify mRNA transcripts in individual cells allows analysis of polygenic activity such as the simultaneous transcription of more than one member of the var multigene family in Plasmodium falciparum infected erythrocytes 1. The technique is adaptable and can be used on different types of genes, cells and organisms.

单细胞基因转录的RNA荧光分析<em在原位</em&gt;杂交（FISH）

Adhesion of Plasmodium falciparum infected erythrocytes (IE) to human endothelial receptors during malaria infections is mediated by expression of PfEMP1 ...

生物学

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for using FISH to quantify the number of mRNAs in single yeast cells. Cells can be grown in any condition of interest and then fixed and made permeable. Subsequently, multiple single-stranded deoxyoligonucleotides conjugated to fluorescent dyes are used to label and visualize mRNAs. Diffraction-limited fluorescence from single mRNA molecules is quantified using a spot-detection algorithm to identify and count the number of mRNAs per cell. While the more standard quantification methods of northern blots, RT-PCR and gene expression microarrays provide information on average mRNAs in the bulk population, FISH facilitates both the counting and localization of these mRNAs in single cells at single-molecule resolution.

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

This protocol describes an experimental procedure for performing Fluorescence in situ Hybridization (FISH) for counting mRNAs in single cells at single-molecule resolution.

可视化和分析的mRNA分子，使用荧光<em&gt;在原位</em&gt;杂交<em&gt;酿酒酵母（Saccharomyces cerevisiae）</em

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for ...

单分子荧光原位杂交实验中单个大肠埃希菌细胞的转录标记方法

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此视频中的章节

单分子荧光原位杂交 (smFISH) 在发芽酵母营养生长和减数分裂中的分析

单分子荧光原位杂交 (SM-FISH) 在小鼠卵母细胞中 Mrna 的定量和定位

体外和体内实验的位定向诱变-以大肠杆菌rna相互作用为例

单分子水平标记均匀性的全蛋白质定量

单分子鱼的转录活性等位基因单细胞分析

用于单分子 FRET-TIRF 显微镜的长 RNA 的荧光末端标记和封装

用于单分子 FRET-TIRF 显微镜的长 RNA 的荧光末端标记和封装

用于单分子 FRET-TIRF 显微镜的长 RNA 的荧光末端标记和封装

单细胞基因转录的RNA荧光分析

可视化和分析的mRNA分子，使用荧光