Name: tissue-specific mirna expression profiling in mouse heart sections using in situ hybridization
Uploaded: 2018-09-15T15:00:00
Description: Watch this Scientific Journal Video about Tissue-specific miRNA Expression Profiling in Mouse Heart Sections Using In Situ Hybridization at JoVE.com

我们要感谢 Athanasios Papangelis 对手稿的批评性评论。FM 由生物技术和生物科学研究理事会 (BBSRC) 支持;BB/M009424/1)。IP 由美国心脏协会科学家发展补助金 (17SDG33060002) 支持。

本研究的小鼠心脏组织样本是按照相关法律和机构指南获得的, 并经耶鲁大学机构动物护理和使用委员会批准。
1. 解决方案准备
<ol><li>RNase 免费 ddH2o, 用5毫升 0.1% diethylpyrocarbonate (DEPC) 在室温下 (RT) 处理5升的 ddH2o。高压釜 (121 °c) 停用 DEPC。使用 DEPC 处理的 ddH2O 进行下游溶液的制备, 如下所示。 注意: DEPC 是已知的致癌物质, 只有油烟机中的手柄。</l...

<ol>
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miRNA 的成绩单检测可以通过不同的技术, 差异的灵敏度, 特异性和数量的权力。在这里, 我们展示了 miRNA原位杂交与染色的耦合, 并描述了一个协议, 允许同时评估 miRNA 和蛋白质分子的表达水平, 在同一心脏部分。我们首先展示了如何在石蜡嵌段心脏切片上进行挖标低噪声 miRNA 探针原位杂交。接下来, 我们描述如何在同一节上执行染色 cTnT。最后, 我们演示了如何合并生成的颜色和荧光?...

微 rna (miRNAs) 是短的 (18–25核苷酸), 单链, 非编码 rna, 在转录后水平的基因表达负调节作用, 抑制信使 RNA (mRNA) 翻译和/或促进 mRNA 退化1. miRNAs 是从编码或非编码基因的内含子或外显子中转录出来的, 并在细胞核中被 DROSHA、前体 miRNAs (前 miRNAs), 后者为70核苷酸2的短茎环结构。在细胞质出口之后, DICER 将 miRNAs 进一步加工成成熟的 miRNAs, 跨度18–25核苷酸3,4。随后, RNA 诱导的沉默复合体 (RISC) 将这些 miRNAs 作为单链 rna, 允许它们绑定到其目标基因的 3 ' 未翻译区 (3 '-UTR), 以抑制其表达式3,5.
在过去的三年里, 自从他们第一次被发现以来, miRNAs 已经出现, 掌握基因表达的调控者, 他们自己的表达水平被严密控制6。miRNAs 的角色已经在器官发育7、8、9、10、11、12、维持稳态13、14, 以及包括神经15、16、17、18、19、心血管20、自身免疫状况21的疾病情境 ,22, 癌症23,24, 其他25。对 miRNA 表达模式相关性的越来越多的认识, 提出了 miRNA 转录的可靠检测方法的必要性。这些方法包括实时 PCR、微阵列、北印迹、原位杂交等, 它们在灵敏度、特异性和定量能力上各不相同, 主要是由于 miRNA 记录由短而高度同源序列6。
我们最近报告了 miRNA-182 在心肌肥厚26的发展中的重要作用, 这一状况描述了心脏的结构适应反应了高血流动力学要求27,28。心脏肥大的特点是心肌质量的增加, 如果与不良重塑29, 可能导致心力衰竭的风险增加, 一个条件占8.5% 的所有死亡归因于心血管疾病30。
在这里, 我们描述了我们的协议, 结合原位杂交与辛标记 (挖掘) 锁定核酸 (低噪声放大) 探针和染色同时检测 miRNA 和蛋白质分子在小鼠心脏组织切片, 在我们的心肌肥厚模型。

<table>
	<tbody>
		<tr>
			<td>Diethylpyrocarbonate</td>
			<td>Sigma Aldrich</td>
			<td>D5758</td>
			<td>DEPC</td>
		</tr>
		<tr>
			<td>Phosphate buffered saline</td>
			<td>Sigma Aldrich</td>
			<td>P4417</td>
			<td>PBS</td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>American Bioanalytical</td>
			<td>AB02038</td>
			<td>non-ionic surfactant</td>
		</tr>
		<tr>
			<td>Histoclear</td>
			<td>National Diagnostics</td>
			<td>HS-200</td>
			<td></td>
		</tr>
		<tr>
			<td>Proteinase K, recombinant, PCR Grade</td>
			<td>Sigma Aldrich</td>
			<td>3115879001</td>
			<td>ProK</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma Aldrich</td>
			<td>P6148</td>
			<td>PFA</td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>ThermoFisher</td>
			<td>S271</td>
			<td>NaCl</td>
		</tr>
		<tr>
			<td>Magnesium Chloride Hexahydrate</td>
			<td>ThermoFisher</td>
			<td>M33</td>
			<td>MgCl2</td>
		</tr>
		<tr>
			<td>Tris</td>
			<td>Sigma Aldrich</td>
			<td>T6066</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrochloric Acid Solution, 1 N</td>
			<td>ThermoFisher</td>
			<td>SA48</td>
			<td>HCl</td>
		</tr>
		<tr>
			<td>Hydrochloric Acid Solution, 12 N</td>
			<td>ThermoFisher</td>
			<td>S25358</td>
			<td>HCl</td>
		</tr>
		<tr>
			<td>1-Methylimidazole</td>
			<td>Sigma Aldrich</td>
			<td>336092</td>
			<td></td>
		</tr>
		<tr>
			<td>N-(3-Dimethylaminopropyl)-N&#8242;-ethylcarbodiimide hydrochloride</td>
			<td>Sigma Aldrich</td>
			<td>39391</td>
			<td>EDC</td>
		</tr>
		<tr>
			<td>Hydrogen peroxide solution H2O2</td>
			<td>Sigma Aldrich</td>
			<td>216763</td>
			<td>H2O2</td>
		</tr>
		<tr>
			<td>Trisodium citrate dihydrate</td>
			<td>Sigma Aldrich</td>
			<td>S1804</td>
			<td>Sodium Citrate</td>
		</tr>
		<tr>
			<td>miRCURY LNA miRNA ISH Buffer Set (FFPE)</td>
			<td>Qiagen</td>
			<td>339450</td>
			<td>scramble miRNA/U6 snRNA</td>
		</tr>
		<tr>
			<td>miRCURY LNA mmu-miR-182 detection probe</td>
			<td>QIagen</td>
			<td>YD00615701</td>
			<td>5&#39;-DIG and 3&#39;-DIG labelled</td>
		</tr>
		<tr>
			<td>Levamisol hydrochloride</td>
			<td>Sigma Aldrich</td>
			<td>31742</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Sigma Aldrich</td>
			<td>A9647</td>
			<td>BSA</td>
		</tr>
		<tr>
			<td>NBT/BCIP Tablets</td>
			<td>Sigma Aldrich</td>
			<td>11697471001</td>
			<td>NBT-BCIP</td>
		</tr>
		<tr>
			<td>Potassium Chloride</td>
			<td>ThermoFisher</td>
			<td>P217</td>
			<td>KCl</td>
		</tr>
		<tr>
			<td>DAPI solution (1mg/ml)</td>
			<td>ThermoFisher</td>
			<td>62248</td>
			<td>DAPI</td>
		</tr>
		<tr>
			<td>Glass coverslip</td>
			<td>ThermoFisher</td>
			<td>12-545E</td>
			<td>Glass coverslip</td>
		</tr>
		<tr>
			<td>Plastic coverslip</td>
			<td>Grace Bio-Labs</td>
			<td>HS40 22mmX40mmX0.25mm</td>
			<td>RNA-ase free plastic coverslip</td>
		</tr>
		<tr>
			<td>Anti-Digoxigenin-AP, Fab fragments</td>
			<td>Sigma Aldrich</td>
			<td>11093274910</td>
			<td>DIG antibody</td>
		</tr>
		<tr>
			<td>Hydrophobic barrier pen</td>
			<td>Vector Laboratories</td>
			<td>H-4000</td>
			<td>Pap pen</td>
		</tr>
		<tr>
			<td>Anti-Cardiac Troponin T antibody</td>
			<td>Abcam</td>
			<td>ab92546</td>
			<td>cTnT antibody</td>
		</tr>
		<tr>
			<td>Goat anti-Rabbit IgG (H+L) Cross-Absorbed Secondary Antibody, Alexa Fluor 568</td>
			<td>ThermoFisher</td>
			<td>A-11011</td>
			<td>anti-rabbit-568 antibody</td>
		</tr>
		<tr>
			<td>Dako Fluorescence Mounting Medium</td>
			<td>DAKO</td>
			<td>S3023</td>
			<td>mounting medium</td>
		</tr>
		<tr>
			<td>Sheep serum</td>
			<td>Sigma Aldrich</td>
			<td>S3772</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat serum</td>
			<td>Sigma Aldrich</td>
			<td>G9023</td>
			<td></td>
		</tr>
		<tr>
			<td>Deionized Formamide</td>
			<td>American Bioanalytical</td>
			<td>AB00600</td>
			<td></td>
		</tr>
		<tr>
			<td>Hybridization Oven</td>
			<td>ThermoFisher</td>
			<td>UVP HB-1000 Hybridizer</td>
			<td></td>
		</tr>
	</tbody>
</table>

tissue-specific mirna expression profiling in mouse heart sections using in situ hybridization

微 rna (miRNAs) 是单链 RNA 转录, 绑定到信使 rna (基因) 和抑制他们的翻译或促进其退化。到目前为止, miRNAs 已牵连到大量的生物和疾病进程, 这标志着需要可靠的检测方法的 miRNA 记录。在这里, 我们描述了一个详细的协议, 辛标记 (挖掘) 锁定核酸 (低噪声放大) 探针为基础的 miRNA 检测, 结合蛋白染色的小鼠心脏切片。首先, 采用原位杂交技术, 利用探针对控制和心肌肥厚小鼠心脏切片中的 miRNA-182 表达进行鉴别。接下来, 我们进行了染色肌钙蛋白 T (cTnT) 蛋白, 在相同的部分, 共同本地化 miRNA-182 与心肌细胞。使用该协议, 我们能够检测 miRNA-182 通过碱性磷酸酶为基础的比色法, 并通过荧光染色 cTnT。该协议可用于检测任何 miRNA 的表达, 通过挖掘标记的低噪声放大探针和相关蛋白表达的小鼠心脏组织切片。

利用 miRNA 和 U6-snRNA 对小鼠心脏切片进行 miRNA原位杂交, 分别作阴性和阳性对照。阳性染色用蓝色表示, 而阴性染色则是由于缺乏颜色发展 (图 1A-1B)。从控制和 PlGF overexpressing 小鼠的心脏切片评价 miRNA-182 心肌细胞特异表达。在αMHC 启动子下携带 PlGF 转基因的小鼠在6周的转基因活化26中发展心肌肥厚, 继发血管生成?...

Watch this Scientific Journal Video about Tissue-specific miRNA Expression Profiling in Mouse Heart Sections Using In Situ Hybridization at JoVE.com

Tissue-specific miRNA Expression Profiling in Mouse Heart Sections Using In Situ Hybridization

原位杂交法在小鼠心脏切片组织特异 miRNA 表达谱分析中的应用

微 rna (miRNAs) 是短而高度同源的 RNA 序列, 充当信使 rna (基因) 的转录后调节剂。目前 miRNA 检测方法的灵敏度和特异性各不相同。我们描述了一个协议, 结合原位杂交和染色的同时检测 miRNA 和蛋白质分子在小鼠心脏组织切片。

micro-RNAs (miRNAs) are single-stranded RNA transcripts that bind to messenger RNAs (mRNAs) and inhibit their translation or promote their degradation. To ...

这种方法通过提供检测蛋白质和微RNA分子的相对化的工具，有助于回答微RNA领域中的关键问题。该技术的主要优点是，您可以检测来自同一组织部分的蛋白质和微RNA分子。该协议从对滑动安装的组织进行补液开始。在杂交烤箱中，在 60 摄氏度下加热滑梯 10 分钟。石蜡应该明显融化了。然后，将幻灯片孵育在装有市售清除剂的玻璃科普林罐中 10 分钟。这样做两次。接下来，将幻灯片从洗涤转移到洗涤，以补充组织水分。一旦水合，在37摄氏度下孵育蛋白酶K工作溶液中的组织15分钟。要去除蛋白酶K，请用1xPBS清洗幻灯片两次。现在，修复组织。首先，使用疏水笔在滑梯上制作储液罐，在准备工作周围绘制一个防水圈。接下来，在屏障内应用 500 微升 4%PFA 溶液，并在烟罩中孵化幻灯片。要去除固定剂，请将包含 PBS 的 Coplin 罐中的幻灯片在室温下两次清洗五分钟。接下来，在0.13摩尔1-甲基米米达佐尔中清洗幻灯片10分钟。在室温下在烟罩中进行此操作，并进行两次此洗涤。加入EDC固定药，在室温下在烟罩中孵育一小时。用 50 毫升三轮车冲洗幻灯片。为了准备杂交，首先建造一个加湿室。将两片滤光片或纸巾装入盒子底部，用一对一的甲酰胺和1x SSC混合物将纸张弄湿。接下来，在每张幻灯片上应用 200 微升预热杂交溶液，并在 50 摄氏度下孵育加湿的幻灯片室 1 至 3 小时。孵育后，用250微升探针溶液代替杂交溶液，用无RNase盖滑盖住幻灯片。尽量避免将气泡困在盖滑下。现在，小心地用副膜密封腔室，并在低于探头RNA熔化温度约30摄氏度的温度下孵育滑轨。可能需要优化。除了设置正确的孵育温度外，不同的微RNA在不同级别上表达，因此可能需要对探头浓度进行优化，以获得最佳信号。混合后，执行串联洗涤。首先，在 2x SSC 在 50 摄氏度下 5 分钟，或直到盖滑松动。然后，在室温下在 2x SSC 中进行两次洗涤，每次洗涤五分钟。然后，在室温下继续，在 0.2 x SSC 中清洗幻灯片 5 分钟，然后在 PBST 中清洗 5 分钟，最后在 1x PBS 中清洗幻灯片 5 分钟。此时，RNA-RNA杂交是稳定的，不再需要RNA严格的条件。对于DID抗体检测，首先，润润疏水屏障，并应用500微升的阻尼溶液30分钟。在加湿室中孵育室温下的幻灯片。接下来，去除阻断溶液，代之以500微升的DI格抗体溶液。在加湿室中将幻灯片在室温下孵育一小时。然后，使用Coplin罐子，在PBST中清洗幻灯片三次，每次洗涤五分钟。接下来，在预染色溶液中清洗幻灯片五分钟两次。然后，将幻灯片传输到聚丙烯滑动邮件器 jar，并添加 20 毫升 AP 基板溶液。现在，在37摄氏度的温度下孵化滑梯6至24小时，不受光线影响。首次使用 AP 基板时，遵循反应非常重要。每隔两小时，使用显微镜检查幻灯片上的颜色发展，直到确定最佳的孵育时间。要去除多余的 AP 基材，请每次洗涤用 KTBT 溶液清洗幻灯片两次，每次洗涤五分钟，然后用 PBS 清洗一次，洗涤五分钟。要检测 cTNT 抗体，首先，阻断组织一小时。然后，应用200微升的cTNT抗体溶液，在4摄氏度的加湿室中孵育。第二天，在含有PBS的科普林罐中清洗幻灯片五分钟。这样做三次。然后，应用250微升抗兔568抗体溶液，在加湿室中孵育幻灯片一小时。要洗掉多余的二次抗体，在PBS中使用三个五分钟的洗涤。然后，如果需要，应用 250 微升的 DAPI 工作解决方案，并孵育幻灯片一分钟，防止光线。要去除多余的 DAPI 污渍，请使用两次五分钟 PBS 洗涤，洗涤后，安装幻灯片。MicroRNA原位杂交在小鼠心脏部分进行了优化，使用炒微RNA进行负对，以及U6 snRNA作为正对照。在对照组和PlGF过度表达小鼠的心脏部分评估了微RNA-182的心肌细胞特异性表达。小鼠在α-MHC启动子下携带PlGF转基因，并在转基因激活后六周内出现心脏肥大，继发性增加血管生成。染色协议显示，在PlGF小鼠心脏中，microRNA-182的表达增加。接下来，要确定哪些细胞类型表示微RNA-182，相同的部分被免疫区为cTNT。也使用了DAPI染色。在对照组和PlGF小鼠心脏部分，在CTNT阳性心肌细胞的核隔间中均发现微RNA-182。看完这段视频后，您应该对如何进行微RNASe杂交，然后是蛋白质免疫保持有一个良好的理解。在尝试此过程时，记住在第一天的所有步骤中保持无 RNAse 条件非常重要。按照这个程序，其他方法，如西方印迹在实时PCR可以执行回答的问题，如，这种特殊的微RNA在不同组织中的相对表达水平，或这种特定的蛋白质在同一组织部分。不要忘记，使用固定材料（如 PFA 和 ADC）可能非常危险。执行此过程时，应始终采取预防措施，包括戴手套和实验室外套，以及使用烟罩。

Research

JoVE Journal

Genetics

Tissue-specific miRNA Expression Profiling in Mouse Heart Sections Using In Situ Hybridization

In Situ Labeling of Mitochondrial DNA Replication in Drosophila Adult Ovaries by EdU Staining

In Situ Labeling of Mitochondrial DNA Replication in Drosophila Adult Ovaries by EdU Staining

在 果蝇卵巢成人线粒体DNA复制的原位标记埃杜染色

The mitochondrial genome is inherited exclusively through the maternal line. Understanding of how the mitochondrion and its genome are proliferated and ...

科研

遗传学

Parallel Measurement of Circadian Clock Gene Expression and Hormone Secretion in Human Primary Cell Cultures

在人原代细胞培养生物钟基因表达及荷尔蒙分泌的并行测量

Circadian clocks are functional in all light-sensitive organisms, allowing for an adaptation to the external world by anticipating daily environmental ...

Optogenetic Random Mutagenesis Using Histone-miniSOG in C. elegans

Optogenetic Random Mutagenesis Using Histone-miniSOG in C. elegans

光遗传学随机突变用组蛋白miniSOG在<em&gt;℃。线虫</em

Forward genetic screening in model organisms is the workhorse to discover functionally important genes and pathways in many biological processes. In most ...

Detection of Inter-chromosomal Stable Aberrations by Multiple Fluorescence In Situ Hybridization (mFISH) and Spectral Karyotyping (SKY) in Irradiated Mice

Detection of Inter-chromosomal Stable Aberrations by Multiple Fluorescence In Situ Hybridization mFISH and Spectral Karyotyping SKY in Irradiated Mice

通过多色荧光跨染色体畸变稳定的检测<em&gt;原位</em&gt;杂交（渔业部）和光谱核型分析（SKY）的照射小鼠

Ionizing radiation (IR) induces numerous stable and unstable chromosomal aberrations. Unstable aberrations, where chromosome morphology is substantially ...

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

单细胞基因表达谱使用流式细胞仪和qPCR内部标准

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

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

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

高分辨率荧光原位杂交在果蝇胚胎和组织中使用 Tyramide 信号放大

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

Profiling DNA Replication Timing Using Zebrafish as an In Vivo Model System

Profiling DNA Replication Timing Using Zebrafish as an In Vivo Model System

使用斑马鱼作为体内模型系统分析 DNA 复制定时

DNA replication timing is an important cellular characteristic, exhibiting significant relationships with chromatin structure, transcription, and DNA ...

Visualization of Microbiota in Tick Guts by Whole-mount In Situ Hybridization

Visualization of Microbiota in Tick Guts by Whole-mount In Situ Hybridization

全挂载原位杂交法可视化蜱内脏微生物群

Infectious diseases transmitted by arthropod vectors continue to pose a significant threat to human health worldwide. The pathogens causing these ...

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

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) 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 ...

Quantitative Analysis of Alternative Pre-mRNA Splicing in Mouse Brain Sections Using RNA In Situ Hybridization Assay

Quantitative Analysis of Alternative Pre-mRNA Splicing in Mouse Brain Sections Using RNA In Situ Hybridization Assay

RNA原位杂交法定量分析小鼠脑切片中的替代前 mRNA 拼接方法

Alternative splicing (AS) occurs in more than 90% of human genes. The expression pattern of an alternatively spliced exon is often regulated in a cell ...