Name: detection of micrornas in microglia by real-time pcr in normal cns and during neuroinflammation
Uploaded: 2012-07-23T13:00:00
Description: Watch this Scientific Journal Video about Detection of MicroRNAs in Microglia by Real-time PCR in Normal CNS and During Neuroinflammation Video at JoVE.com

काम NIH NS071039-01A1 अनुसंधान अनुदान R01 द्वारा समर्थित किया गया.

1. Microglia की सामान्य सीएनएस से अलगाव Microglia के अलगाव के रूप में 3 संशोधनों के साथ पहले से वर्णित किया जाता है. <ol><li> कैंची, संदंश, 27g सुई, 15 मिलीलीटर Dounce homogenizer (Teflon / कांच), 40 माइक्रोन नायलॉन सेल झरनी, 40% और 70% सम...

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	<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>

हाल ही में महत्वपूर्ण ब्याज microglia और इन कोशिकाओं के neuroinflammation और neurodegeneration के साथ जुड़े कई विकृतियों में भागीदारी के लिए दिया गया था. इसके अलावा, प्रतिरक्षा और कैंसर कोशिकाओं के भेदभाव में microRNAs की भूमिका में नाट?...

<table border="1"><tbody><tr><td> अभिकर्मक का नाम </td><td> कंपनी </td><td> सूचीपत्र संख्या </td><td> टिप्पणियाँ </td></tr><tr><td> 1X पीबीएस </td><td> GIBCO </td><td> 14,190 </td><td> 7.2-7.4: शुष्क घटकों से प्रयोगशाला, पीएच में तैयार किया जा सकता है </td></tr><tr><td> 10X पीबीएस </td><td> थर्मो वैज्ञानिक </td><td> SH30258.02 </td><td> 7.2-7.4: शुष्क घटकों से प्रयोगशाला, पीएच में तैयार किया जा सकता है </td></tr><tr><td> DMEM, FBS </td><td> GBCO </td><td> +११,९६५, 10,438 </td><td></td></tr><tr><td> miRVana किट </td><td> Invitrogen </td><td> AM1561 </td><td></td></tr><tr><td> Percoll </td><td> सिग्मा </td><td> P4937 - 500 मिलीलीटर </td><td> 100% की 100 मिलीलीटर Percoll 10X पीबीएस के 10 मिलीग्राम और सिग्मा से Percoll के 90 मिलीग्राम के मिश्रण से तैयार किया जाता है. 70% का 50 मिलीलीटर Percoll35 100% Percoll के मिलीलीटर का मिश्रण द्वारा तैयार और DMEM के 15 मिलीलीटर 40% का 50 मिलीलीटर Percoll 100% Percoll का मिश्रण 20 मिलीग्राम और 30 मिलीलीटर 1X पीबीएस द्वारा तैयार की है </td></tr><tr><td> 35-55 पेप्टाइड MOG </td><td> AnaSpec इंक </td><td> 60,130-5 </td><td></td></tr><tr><td> CFA </td><td> DIFCO </td><td> 263,810 </td><td></td></tr><tr><td> पर्टुसिस विष </td><td> सिग्मा </td><td> P7208 </td><td></td></tr><tr><td> FCR एंटीबॉडी अवरुद्ध </td><td> बी.डी. बायोसाइंसेज </td><td> 553,142 </td><td> क्लोन 2.4G2 </td></tr><tr><td> विरोधी CD11b पीई मैब </td><td> बी.डी. बायोसाइंसेज </td><td> +५५३३११ </td><td> विभिन्न fluorophores (AF488 जैसे) के साथ प्रयोग किया जा सकता है </td></tr><tr><td> विरोधी CD45-APC Cy7 Mabs </td><td> Biolegend </td><td> +१०३११६ </td><td> विभिन्न fluorophores (जैसे APC) के साथ प्रयोग किया जा सकता है </td></tr><tr><td> Paraformaldehyde (16%) </td><td> ईएमएस इंक </td><td> 15,710 </td><td> 1X पीबीएस में 1:15 पतला </td></tr><tr><td> जेल tbe यूरिया 15% </td><td> जीवन टेक्नोलॉजीज इंक </td><td> EC68855BOX </td><td></td></tr><tr><td> TaqMan MicroRNA रिवर्स प्रतिलेखन किट </td><td> जीवन टेक्नोलॉजीज इंक </td><td> 4366596 </td><td></td></tr><tr><td> TaqMan यूनिवर्सल पीसीआर मास्टर मिक्स </td><td> जीवन टेक्नोलॉजीज इंक </td><td> 4304437 </td><td></td></tr><tr><td> TaqMan MicroRNA MMU - मीर 124a Assays </td><td> जीवन टेक्नोलॉजीज इंक </td><td> 4427975 001182 आईडी </td><td></td></tr><tr><td> TaqMan MicroRNA snoRNA-55 Assays </td><td> जीवन टेक्नोलॉजीज इंक </td><td> 4427975 001228 आईडी </td><td></td></tr><tr><td> Nuclease - मुक्त पानी </td><td> जीवन टेक्नोलॉजीज इंक </td><td> AM9937 </td><td> Nuclease - मुक्त पानी </td></tr><tr><td> 384 अच्छी तरह से स्पष्ट ऑप्टिकल प्रतिक्रिया प्लेट </tघ&gt; <td> जीवन टेक्नोलॉजीज इंक </td><td> 4309849 </td><td> अन्य प्लेट (जैसे 96 अच्छी तरह से थाली) के साथ विभिन्न वास्तविक समय पीसीआर उपकरणों में प्रतिस्थापित किया जा सकता है </td></tr><tr><td> Dounce homogenizer (15 मिलीलीटर) </td><td> Wheaton साइंस उत्पाद. </td><td> +३,५८,०४४ </td><td> / Teflon कांच है </td></tr><tr><td> 40 μ नायलॉन सेल झरनी </td><td> बाज़ </td><td> 352,340 </td><td></td></tr><tr><td> बिग अपकेंद्रित्र </td><td> Sorval </td><td> आरटी 6000B </td><td> रोटर व्यास 18.5 सेमी </td></tr><tr><td> लघु अपकेंद्रित्र </td><td> Eppendorf </td><td> 5415 आर </td><td> रोटर व्यास 6.5 सेमी </td></tr><tr><td> वास्तविक समय पीसीआर साधन </td><td> जीवन टेक्नोलॉजीज इंक </td><td> एबी 7900 हिंदुस्तान टाइम्स </td><td> अन्य उपकरणों के साथ प्रतिस्थापित किया जा सकता है </td></tr><tr><td> प्रवाह cytometer </td><td> बी.डी. Biosciencess </td><td> एलएसआर द्वितीय </td><td> अन्य उपकरणों के साथ प्रतिस्थापित किया जा सकता है </p&gt; </tr><tr><td> FACS </td><td> बी.डी. बायोसाइंसेज </td><td> FACSAria </td><td> अन्य उपकरणों के साथ प्रतिस्थापित किया जा सकता है </td></tr><tr><td> Nanodrop स्पेक्ट्रोफोटोमीटर </td><td> थर्मो वैज्ञानिक </td><td> Nanodrop 1000 </td><td></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 Video at JoVE.com

Detection of MicroRNAs in Microglia by Real-time PCR in Normal CNS and During Neuroinflammation Video (Video) | JoVE

Microglia मैक्रोफेज है कि केंद्रीय तंत्रिका तंत्र की रक्षा और प्रतिरक्षा निगरानी की पहली पंक्ति प्रदान निवास कर रहे हैं. MicroRNAs विनियामक अणुओं है कि सक्रियण और मैक्रोफेज के भेदभाव सहित कई शारीरिक प्रक्रियाओं में एक महत्वपूर्ण भूमिका निभा रहे हैं. इस अनुच्छेद में, हम microglia में microRNAs की माप के लिए विधि का वर्णन करता है.

Microglia में microRNAs की सामान्य सीएनएस में वास्तविक समय पीसीआर द्वारा और Neuroinflammation दौरान जांच

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 ...

Research

JoVE Journal

Immunology and Infection

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

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संवहनी और लाइव चूहे में thrombus गठन सूजन के दौरान सक्रिय अन्तःस्तर पर heterotypic प्लेटलेट न्युट्रोफिल बातचीत की वास्तविक समय इमेजिंग

Interaction of activated platelets and leukocytes (mainly neutrophils) on the activated endothelium mediates thrombosis and vascular inflammation.1,2 ...

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Microglia are the resident macrophage-like cells of the central nervous system (CNS) and, as such, have critically important roles in physiological and ...

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During inflammation, leukocytes leave the circulation and cross the endothelium to fight invading pathogens in underlying tissues. This process is known ...

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Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy Video (Video) | JoVE

में माइलॉयड कोशिकाओं गतिशीलता की वास्तविक समय इमेजिंग<em&gt; Apc<sup&gt; न्यूनतम / +</sup</em&gt; ट्यूमर आंतों डिस्क confocal माइक्रोस्कोपी कताई द्वारा

Myeloid cells are the most abundant immune cells within tumors and have been shown to promote tumor progression. Modern intravital imaging techniques ...

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टी सेल रिसेप्टर छांटना सर्किलों के एक साथ मात्रा (TRECs) और कश्मीर हटाने पुनर्संयोजन छांटना हलकों (KRECs) वास्तविक समय पीसीआर द्वारा

T-cell receptor excision circles (TRECs) and K-deleting recombination excision circles (KRECs) are circularized DNA elements formed during recombination ...

High-throughput Quantitative Real-time RT-PCR Assay for Determining Expression Profiles of Types I and III Interferon Subtypes

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उच्च throughput मात्रात्मक वास्तविक समय आरटी पीसीआर परख प्रकार मैं और तृतीय इंटरफेरॉन उपप्रकार की अभिव्यक्ति प्रोफाइल निर्धारण के लिए

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रीयल टाइम में anaesthetized चूहों पर intravital माइक्रोस्कोपी द्वारा Mesenteric रगों में न्यूट्रोफिल और monocytes इमेजिंग

Efficient immune response is dependent on rapid mobilization of blood leukocytes to the site of infection or injury. Investigating leukocyte migration in ...

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नेनो पीसीआर द्वारा पशु नमूनों में श्वसन रोगज़नक़ों के उच्च throughput जांच

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