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
概要
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。
Abstract
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。
Introduction
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。
Protocol
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 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…
Representative Results
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?…
Discussion
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?…
開示
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| Diethylpyrocarbonate | Sigma Aldrich | D5758 | DEPC |
| Phosphate buffered saline | Sigma Aldrich | P4417 | PBS |
| Tween-20 | American Bioanalytical | AB02038 | non-ionic surfactant |
| Histoclear | National Diagnostics | HS-200 | |
| Proteinase K, recombinant, PCR Grade | Sigma Aldrich | 3115879001 | ProK |
| Paraformaldehyde | Sigma Aldrich | P6148 | PFA |
| Sodium Chloride | ThermoFisher | S271 | NaCl |
| Magnesium Chloride Hexahydrate | ThermoFisher | M33 | MgCl2 |
| Tris | Sigma Aldrich | T6066 | |
| Hydrochloric Acid Solution, 1 N | ThermoFisher | SA48 | HCl |
| Hydrochloric Acid Solution, 12 N | ThermoFisher | S25358 | HCl |
| 1-Methylimidazole | Sigma Aldrich | 336092 | |
| N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride | Sigma Aldrich | 39391 | EDC |
| Hydrogen peroxide solution H2O2 | Sigma Aldrich | 216763 | H2O2 |
| Trisodium citrate dihydrate | Sigma Aldrich | S1804 | Sodium Citrate |
| miRCURY LNA miRNA ISH Buffer Set (FFPE) | Qiagen | 339450 | scramble miRNA/U6 snRNA |
| miRCURY LNA mmu-miR-182 detection probe | QIagen | YD00615701 | 5'-DIG and 3'-DIG labelled |
| Levamisol hydrochloride | Sigma Aldrich | 31742 | |
| Bovine Serum Albumin | Sigma Aldrich | A9647 | BSA |
| NBT/BCIP Tablets | Sigma Aldrich | 11697471001 | NBT-BCIP |
| Potassium Chloride | ThermoFisher | P217 | KCl |
| DAPI solution (1mg/ml) | ThermoFisher | 62248 | DAPI |
| Glass coverslip | ThermoFisher | 12-545E | Glass coverslip |
| Plastic coverslip | Grace Bio-Labs | HS40 22mmX40mmX0.25mm | RNA-ase free plastic coverslip |
| Anti-Digoxigenin-AP, Fab fragments | Sigma Aldrich | 11093274910 | DIG antibody |
| Hydrophobic barrier pen | Vector Laboratories | H-4000 | Pap pen |
| Anti-Cardiac Troponin T antibody | Abcam | ab92546 | cTnT antibody |
| Goat anti-Rabbit IgG (H+L) Cross-Absorbed Secondary Antibody, Alexa Fluor 568 | ThermoFisher | A-11011 | anti-rabbit-568 antibody |
| Dako Fluorescence Mounting Medium | DAKO | S3023 | mounting medium |
| Sheep serum | Sigma Aldrich | S3772 | |
| Goat serum | Sigma Aldrich | G9023 | |
| Deionized Formamide | American Bioanalytical | AB00600 | |
| Hybridization Oven | ThermoFisher | UVP HB-1000 Hybridizer |
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