小胶质细胞的小分子RNA检测在正常中枢神经系统的实时PCR技术在神经发炎
Özet
小胶质细胞的居住提供第一线的防御和免疫监视中枢神经系统的巨噬细胞。 microRNA是调节分子，包括巨噬细胞的激活和分化的许多生理过程中发挥了重要作用。在这篇文章中，我们描述了在小胶质细胞的小分子RNA的测量方法。
Abstract
小胶质细胞是驻留在中枢神经系统（CNS）的髓系细胞。这些细胞中发挥了重要作用，在许多疾病的病理与神经发炎，如多发性硬化症（MS）2。在一个正常的中枢神经系统胶质细胞表达巨噬细胞标记CD11b和休息型，如活化标志CD45的表达水平低的表现。在中枢神经系统的病理事件中，成为激活小胶质细胞所确定的上调CD45和其他标记3。在中枢神经系统的小胶质细胞的表型和功能的影响因素没有得到很好的研究。小分子RNA（miRNAs）是一个保守分子（〜22个核苷酸长），在许多正常的生理过程，如细 胞生长和分化的4和5炎症等病症的越来越多的家庭。 miRNA的某些靶基因的表达下调的结合互补序列IR基因和发挥了重要作用，在激活先天免疫细胞，包括巨噬细胞和小胶质细胞7。为了调查定义的小胶质细胞的表型，生物功能，以区别于其他类型的巨噬细胞的小胶质细胞的miRNA介导的途径，它是重要的，特别是在居民中枢神经系统的胶质细胞不同亚群的microRNA表达定量评估。测量在中枢神经系统表达的miRNA的常用方法包括定量PCR技术，从整个神经组织和原位杂交。然而，从整个组织匀浆PCR定量不允许在小胶质细胞的miRNA表达的评估，这代表只有5-15％的神经组织的细胞。 原位杂交允许评估组织切片中的特定类型的细胞的microRNA表达，但这种方法是不完全定量。在这份报告中，我们描述了定量和SENSitive实时PCR检测中的小胶质细胞在正常的中枢神经系统或神经发炎使用实验性自身免疫性脑脊髓炎（EAE）小鼠模型，为MS分离miRNA的方法。所描述的方法将是有益的，在正常的中枢神经系统，或在与各种病症，包括MS，中风，跌打损伤，阿尔茨海默氏症和脑肿瘤相关的神经发炎的小胶质细胞的microRNA表达水平来衡量的。
Protocol
1。从正常中枢神经系统小胶质细胞的分离小胶质细胞的分离以前修改3。 解剖和血流准备工具：剪刀，镊子，10毫升注射器27克针，15毫升Dounce匀浆（铁氟龙/玻璃），40微米尼龙细胞过滤器，40％和70％的Percoll方案，1×PBS。 安乐死小鼠CO 2的相机快窒息（缺氧），并立即转移，手术操作和灌注的实验台。 执行手术操作（用剪刀，镊子，70％?…
Discussion
最近显着的利益，考虑到小胶质细胞，这些细胞参与神经发炎和神经退行性疾病相关的许多病症。此外，在免疫系统和癌细胞分化的microRNA的作用显着增长在过去几年5,10。在非激活或休息的小胶质细胞的表型，在7正常的中枢神经系统的维护，我们曾报道了miR-124的作用，但其他类型的小分子RNA在小胶质细胞的表型和激活状态的作用仍然被发现。这将需要开发专门测量中的小胶质细?…
Açıklamalar
The authors have nothing to disclose.
Acknowledgements
这项工作是由美国国立卫生研究院以R01 NS071039-01A1研究资助支持。
Materials
| Name of the reagent | Company | Catalogue number | Yorumlar |
| 1X PBS | GIBCO | 14190 | Can be prepared from dry components in the laboratory, pH: 7.2-7.4 |
| 10X PBS | Thermo Scientific | SH30258.02 | Can be prepared from dry components in the laboratory, pH: 7.2-7.4 |
| DMEM; FBS | GBCO | 11965; 10438 | |
| miRVana kit | Invitrogen | AM1561 | |
| Percoll | Sigma | P4937-500 ml | 100 ml of 100% Percoll is prepared by mixing 10 ml of 10X PBS and 90 ml of Percoll from Sigma.
50 ml of 70% Percoll is prepared by mixing of 35 ml of 100% Percoll and 15 ml of DMEM 50 ml of 40% Percoll is prepared by mixing 20 ml of 100% Percoll and 30 ml of 1X PBS |
| MOG35-55 peptide | AnaSpec Inc | 60130-5 | |
| CFA | DIFCO | 263810 | |
| Pertussis Toxin | Sigma | P7208 | |
| FcR blocking antibodies | BD Biosciences | 553142 | Clone 2.4G2 |
| Anti-CD11b–PE mAb | BD Biosciences | 553311 | Can be used with different fluorophores (e.g. AF488) |
| Anti-CD45-APC-Cy7 mABs | Biolegend | 103116 | Can be used with different fluorophores (e.g. APC) |
| Paraformaldehyde (16%) | EMS Inc | 15710 | Dilute 1:15 in 1X PBS |
| 15% TBE-Urea Gel | Life Technologies Inc. | EC68855BOX | |
| TaqMan MicroRNA Reverse Transcription Kit | Life Technologies Inc | 4366596 | |
| TaqMan Universal PCR Master Mix | Life Technologies Inc | 4304437 | |
| TaqMan MicroRNA Assays mmu-miR-124a | Life Technologies Inc | 4427975 ID 001182 | |
| TaqMan MicroRNA Assays snoRNA-55 | Life Technologies Inc | 4427975 ID 001228 | |
| Nuclease-free water | Life Technologies Inc | AM9937 | Nuclease-free water |
| 384-well clear optical reaction plate | Life Technologies Inc. | 4309849 | Can be substituted with other plates (e.g. 96 well plate) in different real-time PCR instruments |
| Dounce homogenizer (15 ml) | Wheaton Science Products. | 358044 | Teflon/glass |
| 40 μ nylon cell strainer | Falcon | 352340 | |
| Big centrifuge | Sorval | RT 6000B | Rotor diameter 18.5 cm |
| Small centrifuge | Eppendorf | 5415 R | Rotor diameter 6.5 cm |
| Real-time PCR Instrument | Life Technologies Inc. | AB 7900 HT | Can be substituted with other instruments |
| Flow cytometer | BD Biosciencess | LSR II | Can be substituted with other instruments |
| FACS | BD Biosciences | FACSAria | Can be substituted with other instruments |
| Nanodrop spectrophotometer | Thermo Scientific | Nanodrop 1000 |
Referanslar
- Tremblay, M. E. The Role of Microglia in the Healthy Brain. J. Neurosci. 31, 16064-16069 (2011).
- Graeber, M. B., Li, W., Rodriguez, M. L. Role of microglia in CNS inflammation. FEBS Lett. , (2011).
- Ponomarev, E. D., Shriver, L. P., Maresz, K., Dittel, B. N. Microglial cell activation and proliferation precedes the onset of CNS autoimmunity. J. Neurosci. Res. 81, 374-389 (2005).
- Perruisseau-Carrier, C., Jurga, M., Forraz, N., McGuckin, C. P. miRNAs stem cell reprogramming for neuronal induction and differentiation. Mol. Neurobiol. 43, 215-227 (2011).
- McCoy, C. E. The role of miRNAs in cytokine signaling. Front Biosci. 17, 2161-2171 (2010).
- He, M., Xu, Z., Ding, T., Kuang, D. M., Zheng, L. MicroRNA-155 regulates inflammatory cytokine production in tumor-associated macrophages via targeting C/EBPbeta. Cell Mol. Immunol. 6, 343-352 (2009).
- Ponomarev, E. D., Veremeyko, T., Barteneva, N., Krichevsky, A. M., Weiner, H. L. MicroRNA-124 promotes microglia quiescence and suppresses EAE by deactivating macrophages via the C/EBP-alpha-PU.1 pathway. Nat. Med. 17, 64-70 (2011).
- Basu, S., Campbell, H. M., Dittel, B. N., Ray, A. Purification of Specific Cell Population by Fluorescence Activated Cell Sorting (FACS). J. Vis. Exp. (41), e1546-3791 (2010).
- Gabriely, G. MicroRNA 21 promotes glioma invasion by targeting matrix metalloproteinase regulators. Mol. Cell Biol. 28, 5369-5380 (2008).
- Caffarelli, E., Filetici, P. Epigenetic regulation in cancer development. Front Biosci. 17, 2682-2694 (2011).
- Cardona, A. E., Huang, D., Sasse, M. E., Ransohoff, R. M. Isolation of murine microglial cells for RNA analysis or flow cytometry. Nat. Protoc. 1, 1947-1951 (2006).
- Gordon, R. A simple magnetic separation method for high-yield isolation of pure primary microglia. J. Neurosci. Methods. 194, 287-296 (2011).
- Moltzahn, F., Hunkapiller, N., Mir, A. A., Imbar, T., Blelloch, R. High Throughput MicroRNA Profiling: Optimized Multiplex qRT-PCR at Nanoliter Scale on the Fluidigm Dynamic ArrayTM IFCs. J. Vis. Exp. (54), e2552 (2011).
Bu Makaleden Alıntı Yapın
Veremeyko, T., Starossom, S., Weiner, H. L., Ponomarev, E. D. Detection of MicroRNAs in Microglia by Real-time PCR in Normal CNS and During Neuroinflammation. J. Vis. Exp. (65), e4097, doi:10.3791/4097 (2012).