我们感谢安妮多德森和斯蒂芬妮的建议如何优化 smFISH 协议, 泽维尔 Darzacq 为帮助与分析平台, 海盐黄提出的统计分析建议。这项工作得到了来自3月的钱 (5 FY15-99), 皮尤慈善信托基金 (00027344), 达蒙罗扬癌症研究基金会 (35-15) 和格伦基金会的资助, EÜ, 和 NSF 研究生研究金赠款号。DGE-1106400 到 JC。

注意: 此协议中使用的所有缓冲区和媒体都列在表 1中。试剂的供应商信息列在物料表中。
1天/天 1-2:
注意: 对于植物培养, 在首选培养基中, 将细胞生长到直径为0.4 到0.6 的 OD600 。对于减数分裂培养, 诱导细胞进行减数分裂使用首选方法 (通常培养在 OD600 1.85)。
1. 样品的固定和消化
<ol><li>将3% 个甲醛中的单元格的总直径约为3.5 个600 。<ol>
<li>对于营养培养, 在15毫升圆锥管中加入5.52 毫升的培养基, 480 µL 37% 甲醛。</li>
		<li>对于减数分裂培养, 在2毫升离心管中加入1840µL 的160µL 37% 甲醛的培养。反转 ~ 5 次混合。 注意: 甲醛是有毒的。根据制度规定处理和处分。</li>
	</ol>
</li>
	<li>把管子放在滚筒上, 室温下20分钟。对于减数分裂样品, 在室温下固定20分钟后, 继续固定在4˚C, 旋转。 注意: 隔夜固定提高了 smFISH 的重现性和质量。应优化固定时间。</li>
	<li>虽然样品是固定的, 解冻200毫米钒 Ribonucleoside 复合物 (VRC) 在65˚C 至少10分钟。</li>
	<li>在15毫升管中准备消化大师组合: 1 样品, 混合425µL 的缓冲 B 与40µL 200 毫米 VRC (温暖到65˚C);对于5个样品, 混合2125µL 的缓冲 B 与200µL 200 毫米 VRC (温暖到65˚C)。漩涡 ~ 5 s, 以充分并用重悬 VRC 之前添加到主混合, 这将出现浅褐色绿色后, VRC 添加。 注: 在消化过程中加入 VRC 提高了 smFISH 结果的一致性, 有可能通过抑制 zymolyase 混合物引入的核酸酶污染物, 从粗萃取物中提纯。应优化此步骤所需的 VRC 量。</li>
	<li>对于营养样品, 离心管在〜 1057 x g 3 分钟。对于减数分裂样品 (在隔夜固定后), 离心机在 2.1万 x g 1.5 分钟醒酒或吸入上清到甲醛废料。 注意: 所有的离心步骤都是在室温下进行的。</li>
	<li>并用重悬细胞在1.5 毫升的冷缓冲 B 通过吹打上下或翻转管和弹搅拌混合。泥沙后将植物标本转移到2毫升管。</li>
	<li>离心机在 2.1万 x g 的1.5 分钟. 通过真空吸入或吹打去除液体的大部分, 留下100µL。</li>
	<li>并用重悬1.5 毫升的冷缓冲 B 中的细胞。</li>
	<li>离心机在 2.1万 x g 的1.5 分钟. 通过真空吸入或吹打去除液体的大部分, 留下100µL。</li>
	<li>1.5 毫升冷缓冲器中的并用重悬细胞 b. 离心机在 2.1万 x g 1.5 分钟内完全通过真空或吹打吸入液体。</li>
	<li>并用重悬细胞在425µL 消化大师混合, 并简要涡旋到并用重悬。添加5µL 100T 10 毫克/毫升 zymolyase 每管。 注: 在加入管之前, zymolyase 每一次涡旋。每管单独添加 zymolyase, 而不是添加到主混合。这两个步骤有助于保持管之间的消化一致性, 因为 zymolyase 沉淀迅速。</li>
	<li>漩涡 2-3 s 混合。把管子放在滚筒上, 在30˚C 上消化15-30 分钟。对于营养细胞和早期减数分裂阶段, 通常需要15分钟, 而对于减数分裂前期和减数分裂, 通常需要30分钟。 注: 检查显微镜每5分钟后15分钟, 并停止消化时, 80% 的细胞出现不透明和不屈光。从这一点上来说, 细胞非常脆弱。轻轻地处理细胞, 避免使用真空吸入或涡流.
</li>
	<li>离心管在〜 376 x g 3 分钟, 完全去除液体吹打。</li>
	<li>轻轻地并用重悬细胞与1毫升缓冲 B 由吹打上下1-2 次混合。</li>
	<li>离心管在〜 376 x g 3 分钟, 通过吹打去除缓冲 B 液体。在1毫升的70% 乙醇中轻轻并用重悬细胞 (用无 RNase 水稀释)。</li>
	<li>在室温下孵育 3.5-4 小时。</li>
</ol>2. 杂交
<ol><li>将甲酰胺带到室温下 (50 毫升整除, 在水浴中需要30分钟)。 注: 不要打开甲酰胺瓶, 直到瓶子达到室温, 以避免甲酰胺氧化。 注意: 甲酰胺是有毒的。根据制度规定处理和处分。</li>
	<li>在15毫升圆锥管中制备10% 甲酰胺洗涤缓冲器。</li>
	<li>离心管在〜 376 x g 3 分钟, 去除500µL 70% 乙醇的吹打。轻轻吸管向上和向下并用重悬其余的细胞, 然后转移细胞到低粘附管。 注: 使用低粘接管可大大减少后续洗涤过程中的细胞损耗。</li>
	<li>再离心管在〜 376 x g 为3分钟, 除去所有的乙醇由吹打。</li>
	<li>加入1毫升10% 甲酰胺洗涤缓冲液, 轻轻吸管2-3 次, 并用重悬细胞。</li>
	<li>在准备杂交溶液时, 允许细胞在室温下坐20分钟。对于1样品, 混合50µL 的杂交缓冲 (室温度), 5 µL 200 毫米 VRC (温暖到65˚C) 和1µL 的每个探针 (200 nM 决赛)。对于5个样品, 混合250µL 的杂交缓冲 (室温度), 25 µL 200 毫米 VRC (温暖到65˚C) 和5µL 的每个探针 (200 nM 决赛)。<ol>
<li>解冻杂交缓冲 (冻结在-20 ˚C) 到室温前打开管, 以防止甲酰胺氧化。</li>
		<li>加入200毫米 VRC 后, 涡流为5-10 秒, 然后再添加探头, 这是设计和购买商业, 并根据制造商的指示重组。</li>
		<li>如果两个探头共同孵化, 添加1µL 的探针的1:10 稀释 #1 股票, 以及1µL 的1:10 稀释的探针 #2 股票, 做最后稀释1:500 为任一探针。最好的信噪比所需的浓度需要优化 (参见讨论细节)。</li>
	</ol>
</li>
	<li>将样品离心至 376 x 克, 3 分钟. 吹打将上清入危险废物中。</li>
	<li>在每个管中添加至少50µL 的杂交溶液。轻弹管子混合。</li>
	<li>在30˚C 在滚筒上孵育至少16小时在黑暗中。</li>
</ol>2天/天3
3. 洗涤和成像
<ol><li>把甲酰胺带到室温。</li>
	<li>在15毫升圆锥管中制备10% 甲酰胺洗涤缓冲器 (FWB)。</li>
	<li>从滚筒滚筒上取下管子, 将它们放到箔覆盖的盒子里, 以防光线。</li>
	<li>将样品离心至 376 x 克, 3 分钟. 吹打将上清入危险废物中。</li>
	<li>并用重悬在1毫升 10% FWB 通过轻轻吹打上下2-3 次。</li>
	<li>在箔覆盖盒中孵育30˚C 30 分钟 (不旋转)。</li>
	<li>将样品离心至 376 x 克, 3 分钟. 吹打清除上清至危险废物, 并留下50µL。</li>
	<li>同时, 在15毫升圆锥管中制备 DAPI/FWB: 1 例, 混合1000µL 10% 甲酰胺洗涤缓冲器, 1 毫克µL DAPI。对于10个样品, 混合10毫升10% 甲酰胺洗涤缓冲器与10µL 5 毫克/毫升 DAPI。并用重悬在1毫升的 DAPI/FWB 轻轻吹打上下2-3 次。</li>
	<li>在箔覆盖盒中孵育30˚C 30 分钟 (不旋转)。</li>
	<li>在冰上解冻防漂白试剂。</li>
	<li>离心机样品在 ~ 376 x g 为3分钟, 完全移除上清的吹打。如果需要, 离心机再次清除所有上清。</li>
	<li>对于没有立即成像的样本, 并用重悬在50µL 的 GLOX 缓冲区中不带酶的颗粒。吸管上下3-4 次混合。<ol>
<li>把所有的 unimaged 样品放在铝箔覆盖的盒子里4˚C, 直到准备好图像。当准备好图像, 离心样品在〜 376 x g 为2分钟, 并完全清除上清液吹打。并用重悬在 GLOX 与酶如下。</li>
	</ol>
</li>
	<li>对于正在成像的样品, 添加15-20 µL 的 GLOX 缓冲酶。轻轻吸管上下混合。 注: 添加的体积可以根据细胞颗粒的大小而变化。建议用酶 resuspending 15 µL 的 GLOX 缓冲颗粒, 并在显微镜下检查细胞密度。如果细胞过于致密 (细胞在彼此的顶部聚集), 增加额外的 GLOX 缓冲与酶。如果单元格太稀疏, 请离心取样并取出5µL 的缓冲器。</li>
	<li>吸管5µL 到盖玻片 (18 毫米 x 18 毫米, 1), 并将盖玻片放在幻灯片上。</li>
	<li>在放置盖玻片的幻灯片的顶端放置一个实验室擦拭。轻轻按实验室擦拭设置幻灯片 (应该看到液体从所有四边缘盖玻片)。</li>
	<li>把幻灯片转到显微镜室, 在铝箔覆盖的盒子里。</li>
	<li>图像采用宽场荧光显微镜, 高放大倍数 (60-100X) 和高数值孔径。 注: 为了收集 smFISH 探头发射的光子的最大数量, 不推荐共焦显微镜, 因为它们极大地限制了所收集的光量。在这里, 数据收集在一台装有 100X, 1.4 NA 目标, 使用过滤器 CY5 (EX632/22, EM679/34) 成像在1.3 秒, 100% T;TRITC (EX542/27, EM597/45), 1.3 s, 100% T;和 DAPI (EX390/18, EM435/48), 0.05-0. 1 s, 32%-50% T。还应获取一个明亮的字段参考图像。获取15-25 切片的步骤大小为 0.1-0.2 µm, 从完全低于焦点的视野完全以上, 以确保所有的 RNA 斑点被占。</li>
</ol>4. 图像分析
<ol><li>使用已发布的 smFISH 分析工具 (如鱼量化17和独奏曲2) 和自定义程序 (根据实验的确切要求) 分析 smFISH 数据。 注意: 一个好的分析管道应该允许用户确定一个阈值, 将真实的 RNA 斑点与背景分开, 输出统计数据, 如 X、Y 和 Z (可选) 坐标和每个点的强度、每个单元格中的斑点数。, 并可能将参数作为筛选错误检测的一种方法。</li>
</ol>

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作者没有披露利益冲突。

此处提供的协议来自其他已发布的 smFISH 协议2、3、21、22、23, 并专门针对酵母应变背景 SK1 进行了优化。优化后的参数包括细胞数、固定时间、离心速度和持续时间、消化时间、消化缓冲、探针浓度和杂交缓冲。其他参数, 如杂交温度和持续时间没有优化。在本节中, 我们分享了一些注释, 它将有助于使本议定书适应任何酵母菌的背景和感兴趣的生长条件。
发芽酵母细胞壁是在 smFISH 中获得高质量的图像的主要挑战, 因为细胞壁防止探针穿透。zymolyase 细胞壁的不完全消化导致探针的低效杂交和信号中细胞间的高细胞变异性。然而, 过度消化可能使细胞过于脆弱, 导致在洗涤步骤和细胞爆裂过程中显着细胞损失在幻灯片准备成像。因此, 优化消化时间是至关重要的。我们建议通过在不同的时间内消化相同的样品来进行 smFISH 试验。在我们的情况下, 我们得到了最好的 smFISH 数据, 如果我们停止消化时, 80% 的细胞变得不屈光。通常, 在相同的生长条件下, 不同基因突变株可以用相似的时间来消化。然而, 在发芽酵母的不同生长条件和减数分裂的不同阶段, 消化时间不同。一旦确定了时间, smFISH 的质量就会重现。注意, 对于有缺陷细胞壁合成和/或成分的突变株, 消化可能在15分钟以内完成. 使用不同批次的 zymolyase 也可能略微改变消化时间。
为了达到高信噪比, 需要最佳探针浓度。我们在商业上设计和购买我们的 smFISH 探头。好的探针 (通常是20市场) 应该有一个百分比的 GC 范围从35% 到 45%, 最小间距2基对探头之间, 低交叉反应性。我们使用了一个基于 web 的探头设计器从制造商生成一个探针列表, 如果有足够的探头可以选择, 我们将使用在酿酒基因组数据库中的爆破算法, 以消除超过17基对重叠的探头与其他基因组区域。我们为探测集选择了更 photostable 的荧光染料 (CAL 590), 该探测器组退火NDC80luti (CF 590) 的唯一区域, 因为在此集合中, 满足上述所有条件 (20 探针) 的探头数量低于制造商推荐的最小编号 (25 探头)。在根据制造商的指示重组探头解决方案后, 我们对库存溶液进行了1:10 稀释, 并将稀释溶液储存在 5-µL 整除数-20 ˚C。每个整除只使用一次。为了优化, 我们应该从1:10 稀释溶液中进行串行稀释, 并测试浓度产生最佳信噪比。我们建议从原来的股票做一个稀释系列 1:250, 1:500, 1:1000 和1:2000。在我们的例子中, 1:500 稀释因子导致最佳信噪比, 为 CF 590 和 Q 670 探针集。
最佳的固定时间也是成功 smFISH 的关键。我们发现, 夜间固定在4˚C, 而不是固定在室温20分钟, 大大提高了一致性和质量的 smFISH 结果的减数分裂样本。虽然我们没有测试如何过夜固定可能会影响营养样品, 室温固定在这种生长条件下工作良好。因此, 为了优化 smFISH 协议的新菌株或生长条件, 我们建议开始时, 在室温下固定时间和增加的固定时间, 如果遇到高变异性的信号。
与其他已发布的协议3,22,23相比, 我们的协议使用 RNase 抑制剂 VRC 在消化过程中和较高浓度的 VRC 在杂交期间。在这两个步骤中添加 VRC 提高了 smFISH 结果的一致性, 可能是通过更好地保留 RNA 分子对核酸酶活动, 这可能是由 zymolyase 混合 (酶是从粗提取物纯化, 可能含有 RNase污染物)。因此, 我们建议使用我们的协议中列出的 VRC 量, 或者更高浓度的 VRC 进行优化。
在缓冲 B 和甲酰胺洗涤缓冲液的洗涤步骤中, 细胞的很大一部分可以丢失。为了减少细胞损耗, 可以提高离心速度。在我们的情况下, 使用高速 (2.1万 x g) 颗粒我们的样品在缓冲 B 洗涤明显减少细胞损失。然而, zymolyase 消化后细胞变得非常脆弱, 所以不建议改变离心速度。相反, 我们建议使用来自美国科学的低粘附管, 这大大有助于在洗涤过程中, 在甲酰胺洗涤缓冲细胞颗粒。总的来说, 我们的协议可以持续地生成一层足够致密的细胞来进行高效成像。通常, 7 的视野应该产生 > 130 细胞适合量化。
最后, 确定图像分析所需的最佳参数和输出是很重要的。为了检测 smFISH 点, 发布的分析程序通常用高斯内核过滤原始图像, 以删除背景信号, 并要求用户确定每组图像的信噪比,2,17。不幸的是, 目前还没有一个单一的标准来确定正确的参数, 因此需要对这些不同设置进行一些经验测试。要设置每个步骤所需的参数, 需要迭代地输入不同的参数集, 并检查从每个集合中获得的结果与几个代表性单元中的手动计数是否匹配。一旦找到一组参数, 它可以用于大多数的图像, 尽管不同的生长条件和遗传背景。
此外, 可以测试在量化22之前是否可以执行 smFISH 图像的最大强度投影。这一步简化了光斑检测算法, 并大大减少了成像时间, 但在一些潜在有用的信息, 如个别斑点, 如其亚细胞定位的成本。在我们的例子中, 由NDC80基因产生的 mRNA 分子数量足够低, 这一处理后斑点很好地分离 (在萌芽的酵母3,7) 的成绩单上并不少见。在定位分析至关重要的情况下, 分析管道需要确定每个 smFISH 点在每个通道中的位置, 以评估定位。根据所要求的具体问题, 其他信息, 如每个点的强度可能也需要从管道中提取, 以便进一步分析。优化协议和图像分析管线的关键步骤, 对于获取高品质的 smFISH 数据, 研究感兴趣的问题至关重要。

动态调控基因表达推动生物体的发展, 以及它对环境压力、感染和新陈代谢变化的反应。关注转录调控的研究依赖于测量 RNA 丰度的技术。其中一种方法, 称为单分子荧光原位杂交 (smFISH), 用于检测单个细胞中的单个 RNA 分子1,2。该方法允许测量基因表达中细胞对细胞的变异性和细胞内 RNA 定位的测定。
在最常用的 smFISH 技术中, 对单个 rna 分子的检测需要多个短 DNA 探针 (通常为 48 20 探针), 它们是与目标 RNA 互补的, 并与同一荧光染料相结合。单一荧光探针的结合导致微弱信号, 但所有探针的集合信号是健壮的。这一特性大大提高了信噪比, 因为即使一个探针可以显示出目标绑定, 这种信号与目标 RNA 分子3相比, 预计将非常微弱。在检测误差范围内, RNA 分子的数量可以在不同的生长条件和突变体之间进行计数和比较。
自第一次开发以来, smFISH 已适应研究基因表达式的各个方面4, 如转录伸长1,2, 拼接5,6, 转录爆裂7,8,9, 胞内位基因表达式10,11,12, RNA 本地化13,14,15。最近, 我们用这种方法研究了同一基因的两个转录亚型的表达式, 其中短转录异构体 (NDC80ORF) 具有与长异构体共享的整个序列 (NDC80luti)16。为了唯一地识别两个 mRNA 亚型, 我们使用了两个探针集: 一组特定于NDC80luti的唯一序列, 另一组与另一个荧光染料结合, 绑定到两个亚型的共同区域。NDC80luti rna 被标识为具有两个荧光信号的 colocalized 点, 而NDC80ORF RNA 是仅包含来自公共探测器集的信号的一个。由于NDC80ORF 成绩单的数量是用 "减法" 方法计算的, 因此有必要提高探针杂交的效率和高信噪比, 以自信地识别 smFISH 点并减少误差。传播。
本文介绍了一种优化的协议, 以执行 smFISH 在萌芽酵母酿酒酵母。在该协议中, 对 smFISH 实验中的细胞数、固定时间、消化时间、消化缓冲、探针浓度、杂交缓冲等进行了优化, SK1 了在营养的情况下,酵母的应变背景。生长或减数分裂。然而, 我们也在这篇手稿中注意到: (1) 在图像采集后检查 smFISH 数据质量的方法, (2) 协议中可能需要对不同应变背景和生长条件进行额外优化的步骤。

<table><tbody><tr><td>BSA, RNase-free (50 mg/mL)</td><td>Ambion</td><td>AM2616</td><td>Store at -20 &amp;deg;C.</td></tr><tr><td>Catalase</td><td>Sigma</td><td>C3515</td><td>Store at 4 &amp;deg;C for short-term. Vortex before use.</td></tr><tr><td>DAPI</td><td>Sigma</td><td>D9564</td><td>Store at -20 &amp;deg;C after reconstitution in water. Protect from light.</td></tr><tr><td>50% Dextran Sulfate</td><td>Milli Pore</td><td>S4030</td><td>Store at room temperature. Very viscous liquid. Handle with patience.</td></tr><tr><td>&lt;em&gt;E. coli&lt;/em&gt; tRNA</td><td>Sigma</td><td>R4251</td><td>Store at -20 &amp;deg;C in aliquots after reconstitution in water.</td></tr><tr><td>Ethanol (100%, 200 proof)</td><td>various</td><td></td><td>Flammable.</td></tr><tr><td>37% Formaldehyde</td><td>Fisher</td><td>F79-500</td><td>Store at room temperature. Toxic. Use and dispose with caution.</td></tr><tr><td>Formamide</td><td>Ambion</td><td>AM9342</td><td>Store at 4 &amp;deg;C. Open after the temperature equilibrates to room temperature in order to prevent oxidation. Toxic. Use and dispose with caution.&amp;nbsp;</td></tr><tr><td>Glucose</td><td>various</td><td></td><td>Store at 4 &amp;deg;C after dissolving in nuclease-free water.</td></tr><tr><td>Glucose oxidase</td><td>Sigma</td><td>G2133</td><td>Store at -20 &amp;deg;C in aliquots after reconstitution in 50 mM NaOAc, pH 5.</td></tr><tr><td>Nuclease-free water</td><td>Ambion</td><td>AM9932</td><td>Store at room temperature.</td></tr><tr><td>Potassium phosphate (monobasic and dibasic)</td><td>various</td><td></td><td>Store at room temperature.</td></tr><tr><td>Probe library, diluted in TE, pH 8.0&amp;nbsp;</td><td>Biosearch Technologies</td><td></td><td>Store at -20 &amp;deg;C in aliquots (5 &amp;micro;L) after reconstitution in TE pH 8.0, following the instructions from the manufacturer. Protect from light.&amp;nbsp;</td></tr><tr><td>Sorbitol</td><td>various</td><td></td><td>Store at room temperature.</td></tr><tr><td>20X SSC</td><td>Ambion</td><td>AM9763</td><td>Store at room temperature.</td></tr><tr><td>TE, pH 8.0</td><td>Ambion</td><td>AM9849</td><td>Store at room temperature.</td></tr><tr><td>1 M Tris, pH 8.0</td><td>Ambion</td><td>AM9856</td><td>Store at room temperature.</td></tr><tr><td>Trolox</td><td>Sigma</td><td>238813</td><td>Store at -20 &amp;deg;C in aliquots.</td></tr><tr><td>200 mM Vanadyl ribonucleoside complex (VRC)</td><td>NEB</td><td>S1402S</td><td>Store at -20 &amp;deg;C in aliquots after reconstitution, following the instructions from the manufacturer.</td></tr><tr><td>Zymolyase 100T</td><td>MP Biomedicals</td><td>08320932</td><td>Store at -20 &amp;deg;C in aliquots after reconstitution in water.</td></tr><tr><td>Low-adhesion tubes</td><td>USA Scientific</td><td>1415-2600</td><td>Store at room temperature.</td></tr></tbody></table>

single molecule fluorescence in situ hybridization (smfish) analysis in budding yeast vegetative growth and meiosis

单分子荧光原位杂交 (smFISH) 是研究单细胞基因表达能力的一种强有力的技术, 因为它能够检测和计数单个 RNA 分子。与深度测序方法互补, smFISH 提供了有关转录丰度的细胞间变异和特定 RNA 亚单位定位的信息。最近, 我们用 smFISH 研究了在萌芽酵母减数分裂过程中基因NDC80的表达, 其中存在两个转录亚型, 短转录异构体具有与长异构体共享的整个序列。为了自信地识别每个转录异构体, 我们优化了已知的 smFISH 协议, 并获得了在酵母减数分裂过程中获得的样品的 smFISH 数据的高一致性和质量。在这里, 我们描述了这个优化的协议, 我们用来确定是否获得高质量的 smFISH 数据的标准, 以及在其他酵母菌株和生长条件下实现此协议的一些提示。

Watch this Scientific Journal Video about Single Molecule Fluorescence In Situ Hybridization smFISH Analysis in Budding Yeast Vegetative Growth and Meiosis at JoVE.com

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

该单分子荧光原位杂交协议的优化, 以量化的 RNA 分子在萌芽期酵母在营养生长和减数分裂。

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 ...

single-molecule-fluorescence-situ-hybridization-smfish-analysis

Research

JoVE Journal

Genetics

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

19.2K 次观看.  University of California, Berkeley. 该单分子荧光原位杂交协议的优化, 以量化的 RNA 分子在萌芽期酵母在营养生长和减数分裂。

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

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), ...

科研

遗传学

Using Fluorescence In Situ Hybridization (FISH) to Monitor the State of Arm Cohesion in Prometaphase and Metaphase I Drosophila Oocytes

In humans, chromosome segregation errors in oocytes are responsible for the majority of miscarriages and birth defects. Moreover, as women age, their risk of conceiving an aneuploid fetus increases dramatically and this phenomenon is known as the maternal age effect. One requirement for accurate chromosome segregation during the meiotic divisions is maintenance of sister chromatid cohesion during the extended prophase period that oocytes experience. Cytological evidence in both humans and model organisms suggests that meiotic cohesion deteriorates during the aging process. In addition, segregation errors in human oocytes are most prevalent during meiosis I, consistent with premature loss of arm cohesion. The use of model organisms is critical for unraveling the mechanisms that underlie age-dependent loss of cohesion. Drosophila melanogaster offers several advantages for studying the regulation of meiotic cohesion in oocytes. However, until recently, only genetic tests were available to assay for loss of arm cohesion in oocytes of different genotypes or under different experimental conditions. Here, a detailed protocol is provided for using fluorescence in situ hybridization (FISH) to directly visualize defects in arm cohesion in prometaphase I and metaphase I arrested Drosophila oocytes. By generating a FISH probe that hybridizes to the distal arm of the X chromosome and collecting confocal Z stacks, a researcher can visualize the number of individual FISH signals in three dimensions and determine whether sister chromatid arms are separated. The procedure outlined makes it possible to quantify arm cohesion defects in hundreds of Drosophila oocytes. As such, this method provides an important tool for investigating the mechanisms that contribute to cohesion maintenance as well as the factors that lead to its demise during the aging process.

Using Fluorescence In Situ Hybridization FISH to Monitor the State of Arm Cohesion in Prometaphase and Metaphase I Drosophila Oocytes

This manuscript presents a detailed method for generating X-chromosome arm probes and performing fluorescence in situ hybridization (FISH) to examine the state of sister chromatid cohesion in prometaphase and metaphase I arrested Drosophila oocytes. This protocol is suitable for determining whether meiotic arm cohesion is intact or disrupted in different genotypes.

利用荧光原位杂交 (fish) 监测中期和中期 I. 型果蝇卵母细胞的臂部凝聚状态

In humans, chromosome segregation errors in oocytes are responsible for the majority of miscarriages and birth defects. Moreover, as women age, their risk ...

发育生物学

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

Quantitative Live Cell Fluorescence-microscopy Analysis of Fission Yeast

Several microscopy techniques are available today that can detect a specific protein within the cell. During the last decade live cell imaging using fluorochromes like Green Fluorescent Protein (GFP) directly attached to the protein of interest has become increasingly popular 1. Using GFP and similar fluorochromes the subcellular localisations and movements of proteins can be detected in a fluorescent microscope. Moreover, also the subnuclear localisation of a certain region of a chromosome can be studied using this technique. GFP is fused to the Lac Repressor protein (LacR) and ectopically expressed in the cell where tandem repeats of the lacO sequence has been inserted into the region of interest on the chromosome2. The LacR-GFP will bind to the lacO repeats and that area of the genome will be visible as a green dot in the fluorescence microscope. Yeast is especially suited for this type of manipulation since homologous recombination is very efficient and thereby enables targeted integration of the lacO repeats and engineered fusion proteins with GFP 3. Here we describe a quantitative method for live cell analysis of fission yeast. Additional protocols for live cell analysis of fission yeast can be found, for example on how to make a movie of the meiotic chromosomal behaviour 4. In this particular experiment we focus on subnuclear organisation and how it is affected during gene induction. We have labelled a gene cluster, named Chr1, by the introduction of lacO binding sites in the vicinity of the genes. The gene cluster is enriched for genes that are induced early during nitrogen starvation of fission yeast 5. In the strain the nuclear membrane (NM) is labelled by the attachment of mCherry to the NM protein Cut11 giving rise to a red fluorescent signal. The Spindle Pole body (SPB) compound Sid4 is fused to Red Fluorescent Protein (Sid4-mRFP) 6. In vegetatively growing yeast cells the centromeres are always attached to the SPB that is embedded in the NM 7. The SPB is identified as a large round structure in the NM. By imaging before and 20 minutes after depletion of the nitrogen source we can determine the distance between the gene cluster (GFP) and the NM/SPB. The mean or median distances before and after nitrogen depletion are compared and we can thus quantify whether or not there is a shift in subcellular localisation of the gene cluster after nitrogen depletion.

The fission yeast, Schizosaccharomyces pombe, is a good model system to study basic cellular processes. Here we describe a method to perform quantitative live cell analysis of fission yeast. In this particular experiment we focus on organisation of the genome within the cell nucleus, but the method can also be used to study cytosolic factors.

裂殖酵母的活细胞定量荧光显微镜分析

Several microscopy techniques are available today that can detect a specific protein within the cell. During the last decade live cell imaging using ...

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 ...

Combined DNA-RNA Fluorescent In situ Hybridization (FISH) to Study X Chromosome Inactivation in Differentiated Female Mouse Embryonic Stem Cells

Fluorescent in situ hybridization (FISH) is a molecular technique which enables the detection of nucleic acids in cells. DNA FISH is often used in cytogenetics and cancer diagnostics, and can detect aberrations of the genome, which often has important clinical implications. RNA FISH can be used to detect RNA molecules in cells and has provided important insights in regulation of gene expression. Combining DNA and RNA FISH within the same cell is technically challenging, as conditions suitable for DNA FISH might be too harsh for fragile, single stranded RNA molecules. We here present an easily applicable protocol which enables the combined, simultaneous detection of Xist RNA and DNA encoded by the X chromosomes. This combined DNA-RNA FISH protocol can likely be applied to other systems where both RNA and DNA need to be detected.

Combined DNA-RNA Fluorescent In situ Hybridization FISH to Study X Chromosome Inactivation in Differentiated Female Mouse Embryonic Stem Cells

Fluorescent in situ hybridization (FISH) allows the detection of nucleic acids in their native environment within cells. We here describe a protocol for the combined, simultaneous detection of RNA and DNA by means of FISH, which can be used to study X chromosome inactivation in mouse embryonic stem cells.

结合DNA-RNA荧光<em&gt;原位</em&gt;杂交（FISH）研究X染色体失活的分化雌​​性小鼠胚胎干细胞

Fluorescent in situ hybridization (FISH) is a molecular technique which enables the detection of nucleic acids in cells. DNA FISH is often used in ...

Microscopy of Fission Yeast Sexual Lifecycle

The fission yeast Schizosaccharomyces pombe has been an invaluable model system in studying the regulation of the mitotic cell cycle progression, the mechanics of cell division and cell polarity. Furthermore, classical experiments on its sexual reproduction have yielded results pivotal to current understanding of DNA recombination and meiosis. More recent analysis of fission yeast mating has raised interesting questions on extrinsic stimuli response mechanisms, polarized cell growth and cell-cell fusion. To study these topics in detail we have developed a simple protocol for microscopy of the entire sexual lifecycle. The method described here is easily adjusted to study specific mating stages. Briefly, after being grown to exponential phase in a nitrogen-rich medium, cell cultures are shifted to a nitrogen-deprived medium for periods of time suited to the stage of the sexual lifecycle that will be explored. Cells are then mounted on custom, easily built agarose pad chambers for imaging. This approach allows cells to be monitored from the onset of mating to the final formation of spores.

We provide a reproducible basic method for the long-term microscopy of the fission yeast sexual lifecycle. With minor adjustments described, the presented protocol allows research focus on different steps of the reproductive process.

裂殖酵母性生命周期的显微镜

The fission yeast Schizosaccharomyces pombe has been an invaluable model system in studying the regulation of the mitotic cell cycle progression, the ...

Examination of Mitotic and Meiotic Fission Yeast Nuclear Dynamics by Fluorescence Live-cell Microscopy

Live-cell imaging is a microscopy technique used to examine cell and protein dynamics in living cells. This imaging method is not toxic, generally does not interfere with cell physiology, and requires minimal experimental handling. The low levels of technical interference enable researchers to study cells across multiple cycles of mitosis and to observe meiosis from beginning to end. Using fluorescent tags such as Green Fluorescent Protein (GFP) and Red Fluorescent Protein (RFP), researchers can analyze different factors whose functions are important for processes like transcription, DNA replication, cohesion, and segregation. Coupled with data analysis using Fiji (a free, optimized ImageJ version), live-cell imaging offers various ways of assessing protein movement, localization, stability, and timing, as well as nuclear dynamics and chromosome segregation. However, as is the case with other microscopy methods, live-cell imaging is limited by the intrinsic properties of light, which put a limit to the resolution power at high magnifications, and is also sensitive to photobleaching or phototoxicity at high wavelength frequencies. However, with some care, investigators can bypass these physical limitations by carefully choosing the right conditions, strains, and fluorescent markers to allow for the appropriate visualization of mitotic and meiotic events.

Here, we present live-cell imaging which is a non-toxic microscopy method that allows researchers to study protein behavior and nuclear dynamics in living fission yeast cells during mitosis and meiosis.

荧光活细胞显微镜对微孔裂变酵母核动力学的检测

Live-cell imaging is a microscopy technique used to examine cell and protein dynamics in living cells. This imaging method is not toxic, generally does ...

Probing mRNA Kinetics in Space and Time in Escherichia coli using Two-Color Single-Molecule Fluorescence In Situ Hybridization

Single-molecule fluorescence in situ hybridization (smFISH) allows for counting the absolute number of mRNAs in individual cells. Here, we describe an application of smFISH to measure the rates of transcription and mRNA degradation in Escherichia coli. As smFISH is based on fixed cells, we perform smFISH at multiple time points during a time-course experiment, i.e., when cells are undergoing synchronized changes upon induction or repression of gene expression. At each time point, sub-regions of an mRNA are spectrally distinguished to probe transcription elongation and premature termination. The outcome of this protocol also allows for analyzing intracellular localization of mRNAs and heterogeneity in mRNA copy numbers among cells. Using this protocol many samples (~50) can be processed within 8 h, like the amount of time needed for just a few samples. We discuss how to apply this protocol to study the transcription and degradation kinetics of different mRNAs in bacterial cells.

Probing mRNA Kinetics in Space and Time in Escherichia coli using Two-Color Single-Molecule Fluorescence In Situ Hybridization

This protocol describes an application of single-molecule fluorescence in situ hybridization (smFISH) to measure the in vivo kinetics of mRNA synthesis and degradation.

利用两色单分子荧光在原位杂交中Escherichia coli探索大肠杆菌中空间和时间中的 mRNA 动力学

Single-molecule fluorescence in situ hybridization (smFISH) allows for counting the absolute number of mRNAs in individual cells. Here, we describe an ...

Use of Time-Lapse Microscopy and Stage-Specific Nuclear Depletion of Proteins to Study Meiosis in S. cerevisiae

Time-lapse fluorescence microscopy has revolutionized the understanding of meiotic cell-cycle events by providing temporal and spatial data that is often not seen by imaging fixed cells. Budding yeast has proved to be an important model organism to study meiotic chromosome segregation because many meiotic genes are highly conserved. Time-lapse microscopy of meiosis in budding yeast allows the monitoring of different meiotic mutants to show how the mutation disrupts meiotic processes. However, many proteins function at multiple points in meiosis. The use of loss-of-function or meiotic null mutants can therefore disrupt an early process, blocking or disturbing the later process and making it difficult to determine the phenotypes associated with each individual role. To circumvent this challenge, this protocol describes how the proteins can be conditionally depleted from the nucleus at specific stages of meiosis while monitoring meiotic events using time-lapse microscopy. Specifically, this protocol describes how the cells are synchronized in prophase I, how the anchor away technique is used to deplete proteins from the nucleus at specific meiotic stages, and how time-lapse imaging is used to monitor meiotic chromosome segregation. As an example of the usefulness of the technique, the kinetochore protein Ctf19 was depleted from the nucleus at different time points during meiosis, and the number of chromatin masses was analyzed at the end of meiosis II. Overall, this protocol can be adapted to deplete different nuclear proteins from the nucleus while monitoring the meiotic divisions.

Use of Time-Lapse Microscopy and Stage-Specific Nuclear Depletion of Proteins to Study Meiosis in S. cerevisiae

Time-lapse microscopy is a valuable tool for studying meiosis in budding yeast. This protocol describes a method that combines cell-cycle synchronization, time-lapse microscopy, and conditional depletion of a target protein to demonstrate how to study the function of a specific protein during meiotic chromosome segregation.

使用延时显微镜和蛋白质的阶段特异性核耗竭来研究酿酒酵母的减数分裂

Time-lapse fluorescence microscopy has revolutionized the understanding of meiotic cell-cycle events by providing temporal and spatial data that is often ...

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

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