The work was supported by the NIH R01 NS071039-01A1 research grant.

1. Isolation of Microglia from Normal CNS
Isolation of microglia is performed as was described previously with modifications3.
<ol>
 <li>Prepare instruments for dissection and perfusion: scissors, forceps, 10 ml syringe with 27g needle, 15 ml Dounce homogenizer (Teflon/glass), 40 &mu;m nylon cell strainer, 40% and 70% Percoll solutions, 1X PBS.</li>
 <li>Euthanize mice by quick asphyxiation (deprivation of oxygen) in CO2 camera and immediately transfer them to laborator...

<ol>
	<li>Tremblay, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Role+of+Microglia+in+the+Healthy+Brain.">The Role of Microglia in the Healthy Brain.</a> J. Neurosci. 31, 16064-16069 (2011).</li><li>Graeber, M. B., Li, W., Rodriguez, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+microglia+in+CNS+inflammation.">Role of microglia in CNS inflammation.</a> FEBS Lett. , (2011).</li><li>Ponomarev, E. D., Shriver, L. P., Maresz, K., Dittel, B. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglial+cell+activation+and+proliferation+precedes+the+onset+of+CNS+autoimmunity.">Microglial cell activation and proliferation precedes the onset of CNS autoimmunity.</a> J. Neurosci. Res. 81, 374-389 (2005).</li><li>Perruisseau-Carrier, C., Jurga, M., Forraz, N., McGuckin, C. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=miRNAs+stem+cell+reprogramming+for+neuronal+induction+and+differentiation.">miRNAs stem cell reprogramming for neuronal induction and differentiation.</a> Mol. Neurobiol. 43, 215-227 (2011).</li><li>McCoy, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+miRNAs+in+cytokine+signaling.">The role of miRNAs in cytokine signaling.</a> Front Biosci. 17, 2161-2171 (2010).</li><li>He, M., Xu, Z., Ding, T., Kuang, D. M., Zheng, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNA-155+regulates+inflammatory+cytokine+production+in+tumor-associated+macrophages+via+targeting+C/EBPbeta.">MicroRNA-155 regulates inflammatory cytokine production in tumor-associated macrophages via targeting C/EBPbeta.</a> Cell Mol. Immunol. 6, 343-352 (2009).</li><li>Ponomarev, E. D., Veremeyko, T., Barteneva, N., Krichevsky, A. M., Weiner, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNA-124+promotes+microglia+quiescence+and+suppresses+EAE+by+deactivating+macrophages+via+the+C/EBP-alpha-PU.1+pathway.">MicroRNA-124 promotes microglia quiescence and suppresses EAE by deactivating macrophages via the C/EBP-alpha-PU.1 pathway.</a> Nat. Med. 17, 64-70 (2011).</li><li>Basu, S., Campbell, H. M., Dittel, B. N., Ray, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+Specific+Cell+Population+by+Fluorescence+Activated+Cell+Sorting+(FACS).">Purification of Specific Cell Population by Fluorescence Activated Cell Sorting (FACS).</a> J. Vis. Exp. (41), e1546-3791 (2010).</li><li>Gabriely, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNA+21+promotes+glioma+invasion+by+targeting+matrix+metalloproteinase+regulators.">MicroRNA 21 promotes glioma invasion by targeting matrix metalloproteinase regulators.</a> Mol. Cell Biol. 28, 5369-5380 (2008).</li><li>Caffarelli, E., Filetici, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epigenetic+regulation+in+cancer+development.">Epigenetic regulation in cancer development.</a> Front Biosci. 17, 2682-2694 (2011).</li><li>Cardona, A. E., Huang, D., Sasse, M. E., Ransohoff, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+murine+microglial+cells+for+RNA+analysis+or+flow+cytometry.">Isolation of murine microglial cells for RNA analysis or flow cytometry.</a> Nat. Protoc. 1, 1947-1951 (2006).</li><li>Gordon, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+magnetic+separation+method+for+high-yield+isolation+of+pure+primary+microglia.">A simple magnetic separation method for high-yield isolation of pure primary microglia.</a> J. Neurosci. Methods. 194, 287-296 (2011).</li><li>Moltzahn, F., Hunkapiller, N., Mir, A. A., Imbar, T., Blelloch, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+Throughput+MicroRNA+Profiling:+Optimized+Multiplex+qRT-PCR+at+Nanoliter+Scale+on+the+Fluidigm+Dynamic+ArrayTM+IFCs.">High Throughput MicroRNA Profiling: Optimized Multiplex qRT-PCR at Nanoliter Scale on the Fluidigm Dynamic ArrayTM IFCs.</a> J. Vis. Exp. (54), e2552 (2011).</li></ol>

Recently significant interest was given to microglia and involvement of these cells in many pathologies associated with neuroinflammation and neurodegeneration. In addition, the role of microRNAs in the differentiation of immune and cancer cells has dramatically grown over the last several years5,10. We have previously reported the role of miR-124 in the maintenance of non-activated or resting phenotype of microglia in normal CNS7, but the role of the other types of microRNAs in microglia phenotype ...

<table border="1">
<tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td> 1X PBS</td>
 <td> GIBCO</td>
 <td> 14190</td>
 <td>Can be prepared from dry components in the laboratory, pH: 			7.2-7.4</td>
 </tr>
 <tr>
 <td> 10X PBS</td>
 <td> Thermo Scientific</td>
 <td> SH30258.02</td>
 <td>Can be prepared from dry components in the laboratory, pH: 			7.2-7.4</td>
 </tr>
 <tr>
 <td>DMEM; FBS</td>
 <td>GBCO</td>
 <td>11965; 10438</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>miRVana kit</td>
 <td>Invitrogen</td>
 <td>AM1561</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td> Percoll</td>
 <td> Sigma</td>
 <td> P4937-500 ml</td>
 <td>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</td>
 </tr>
 <tr>
 <td>MOG35-55 peptide</td>
 <td>AnaSpec Inc</td>
 <td>60130-5</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>CFA</td>
 <td>DIFCO</td>
 <td>263810</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Pertussis Toxin</td>
 <td>Sigma</td>
 <td>P7208</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>FcR blocking antibodies</td>
 <td>BD Biosciences</td>
 <td>553142</td>
 <td>Clone 2.4G2</td>
 </tr>
 <tr>
 <td> Anti-CD11b-PE mAb</td>
 <td> BD Biosciences</td>
 <td> 553311</td>
 <td>Can be used with different fluorophores (e.g. AF488)</td>
 </tr>
 <tr>
 <td> Anti-CD45-APC-Cy7 mABs</td>
 <td> Biolegend</td>
 <td> 103116</td>
 <td>Can be used with different fluorophores (e.g. APC)</td>
 </tr>
 <tr>
 <td>Paraformaldehyde (16%)</td>
 <td>EMS Inc</td>
 <td>15710</td>
 <td>Dilute 1:15 in 1X PBS</td>
 </tr>
 <tr>
 <td>15% TBE-Urea Gel</td>
 <td>Life Technologies Inc.</td>
 <td>EC68855BOX</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>TaqMan MicroRNA Reverse Transcription Kit</td>
 <td>Life Technologies Inc</td>
 <td>4366596</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>TaqMan Universal PCR Master Mix</td>
 <td>Life Technologies Inc</td>
 <td>4304437</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>TaqMan MicroRNA Assays mmu-miR-124a</td>
 <td>Life Technologies Inc</td>
 <td>4427975 ID 001182</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>TaqMan MicroRNA Assays snoRNA-55</td>
 <td>Life Technologies Inc</td>
 <td>4427975 ID 001228</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Nuclease-free water</td>
 <td>Life Technologies Inc</td>
 <td>AM9937</td>
 <td>Nuclease-free water</td>
 </tr>
 <tr>
 <td> 384-well clear optical reaction plate</td>
 <td> Life Technologies Inc.</td>
 <td> 4309849</td>
 <td>Can be substituted with other plates (e.g. 96 well plate) in 			different real-time PCR instruments</td>
 </tr>
 <tr>
 <td>Dounce homogenizer (15 ml)</td>
 <td>Wheaton Science Products.</td>
 <td> 358044</td>
 <td> Teflon/glass</td>
 </tr>
 <tr>
 <td>40 &mu; nylon cell strainer</td>
 <td>Falcon</td>
 <td>352340</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Big centrifuge</td>
 <td>Sorval</td>
 <td>RT 6000B</td>
 <td>Rotor diameter 18.5 cm</td>
 </tr>
 <tr>
 <td>Small centrifuge</td>
 <td>Eppendorf</td>
 <td>5415 R</td>
 <td>Rotor diameter 6.5 cm</td>
 </tr>
 <tr>
 <td>Real-time PCR Instrument</td>
 <td>Life Technologies Inc.</td>
 <td>AB 7900 HT</td>
 <td>Can be substituted with other instruments</td>
 </tr>
 <tr>
 <td>Flow cytometer</td>
 <td>BD Biosciencess</td>
 <td>LSR II</td>
 <td>Can be substituted with other instruments</td>
 </tr>
 <tr>
 <td>FACS</td>
 <td>BD Biosciences</td>
 <td>FACSAria</td>
 <td>Can be substituted with other instruments</td>
 </tr>
 <tr>
 <td>Nanodrop spectrophotometer</td>
 <td>Thermo Scientific</td>
 <td>Nanodrop 1000</td>
 <td>&nbsp;</td>
 </tr>
 </tbody>
</table>

detection of micrornas in microglia by real-time pcr in normal cns and during neuroinflammation

Microglia are cells of the myeloid lineage that reside in the central nervous system (CNS)1. These cells play an important role in pathologies of many diseases associated with neuroinflammation such as multiple sclerosis (MS)2. Microglia in a normal CNS express macrophage marker CD11b and exhibit a resting phenotype by expressing low levels of activation markers such as CD45. During pathological events in the CNS, microglia become activated as determined by upregulation of CD45 and other markers3. The factors that affect microglia phenotype and functions in the CNS are not well studied. MicroRNAs (miRNAs) are a growing family of conserved molecules (~22 nucleotides long) that are involved in many normal physiological processes such as cell growth and differentiation4 and pathologies such as inflammation5. MiRNAs downregulate the expression of certain target genes by binding complementary sequences of their mRNAs and play an important role in the activation of innate immune cells including macrophages6 and microglia7. In order to investigate miRNA-mediated pathways that define the microglial phenotype, biological function, and to distinguish microglia from other types of macrophages, it is important to quantitatively assess the expression of particular microRNAs in distinct subsets of CNS-resident microglia. Common methods for measuring the expression of miRNAs in the CNS include quantitative PCR from whole neuronal tissue and in situ hybridization. However, quantitative PCR from whole tissue homogenate does not allow the assessment of the expression of miRNA in microglia, which represent only 5-15% of the cells of neuronal tissue. Hybridization in situ allows the assessment of the expression of microRNA in specific cell types in the tissue sections, but this method is not entirely quantitative. In this report we describe a quantitative and sensitive method for the detection of miRNA by real-time PCR in microglia isolated from normal CNS or during neuroinflammation using experimental autoimmune encephalomyelitis (EAE), a mouse model for MS. The described method will be useful to measure the level of expression of microRNAs in microglia in normal CNS or during neuroinflammation associated with various pathologies including MS, stroke, traumatic injury, Alzheimer's disease and brain tumors.

Watch this Scientific Journal Video about Detection of MicroRNAs in Microglia by Real-time PCR in Normal CNS and During Neuroinflammation at JoVE.com

Detection of MicroRNAs in Microglia by Real-time PCR in Normal CNS and During Neuroinflammation

Microglia are resident macrophages that provide the first line of defense and immune surveillance of the central nervous system. MicroRNAs are regulatory molecules that play an important role in many physiological processes including activation and differentiation of macrophages. In this article, we describe the method for measurement of microRNAs in microglia.

Microglia are cells of the myeloid lineage that reside in the central nervous system (CNS)1. These cells play an important role in pathologies of many ...

detection-micrornas-microglia-real-time-pcr-normal-cns-during

Research

JoVE Journal

Immunology and Infection

19.3K visualizações.  Harvard Medical School. Microglia residem macrófagos que fornecem a primeira linha de defesa e vigilância imune do sistema nervoso central. MicroRNAs são moléculas reguladoras, que desempenham um papel importante em muitos processos fisiológicos, incluindo a activação e diferenciação dos macrófagos. Neste artigo, descrevemos o método para a medição de microRNAs em microglia.