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

Arbejdet blev støttet af NIH R01 NS071039-01A1 forskningsbevilling.

1. Isolering af mikroglia fra normale CNS Isolering af mikroglia udføres som beskrevet tidligere med modifikationer 3. <ol><li> Forberede instrumenter til dissektion og perfusion: saks, tænger 10 ml sprøjte med 27g kanyle 15 ml Dounce-homogenisator (Teflon / glas), 40 um nylon cellefilter, 40% og 70% Percoll opløsninger, 1X PBS. </li><li> Afliv mus ved hurtig kvælning (berøvelse af ilt) i CO 2 kamera og straks overføre dem til laboratoriet bænk for kirurgiske ...

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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. 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Nylig væsentlig interesse blev givet til microglia og involvering af disse celler i mange patologier forbundet med neuroinflammation og neurodegeneration. Desuden har rolle microRNA&#39;er i differentieringen af immun-og cancerceller dramatisk dyrket i løbet af de sidste mange år 5,10. Vi har tidligere rapporteret rolle miR-124 i opretholdelse af ikke-aktiverede eller hvilende fænotype af mikroglia i normale CNS 7, men rollen af de andre typer microRNA&#39;er i microglia fænotype og aktivering...

<table border="1"><tbody><tr><td> Navn på reagenset </td><td> Firma </td><td> Katalog nummer </td><td> Kommentarer </td></tr><tr><td> 1X PBS </td><td> GIBCO </td><td> 14190 </td><td> Kan fremstilles ud fra tørre komponenter i laboratoriet, pH: 7,2-7,4 </td></tr><tr><td> 10X PBS </td><td> Thermo Scientific </td><td> SH30258.02 </td><td> Kan fremstilles ud fra tørre komponenter i laboratoriet, pH: 7,2-7,4 </td></tr><tr><td> DMEM; FBS </td><td> GBCO </td><td> 11965; 10438 </td><td></td></tr><tr><td> Mirvana kit </td><td> Invitrogen </td><td> AM1561 </td><td></td></tr><tr><td> Percoll </td><td> Sigma </td><td> P4937-500 ml </td><td> 100 ml 100% Percoll fremstilles ved at blande 10 ml af 10X PBS og 90 ml Percoll fra Sigma. 50 ml 70% Percoll erfremstilles ved blanding af 35 ml 100% Percoll og 15 ml DMEM 50 ml 40% Percoll fremstilles ved at blande 20 ml 100% Percoll og 30 ml 1X PBS </td></tr><tr><td> MOG 35-55 peptid </td><td> Hjælp af Anaspec Inc </td><td> 60130-5 </td><td></td></tr><tr><td> CFA </td><td> DIFCO </td><td> 263810 </td><td></td></tr><tr><td> Pertussis-toxin </td><td> Sigma </td><td> P7208 </td><td></td></tr><tr><td> FcR blokerende antistoffer </td><td> BD Biosciences </td><td> 553142 </td><td> Klon 2.4G2 </td></tr><tr><td> Anti-CD11b - PE mAb </td><td> BD Biosciences </td><td> 553311 </td><td> Kan anvendes med forskellige fluorophorer (f.eks AF488) </td></tr><tr><td> Anti-CD45-APC-Cy7 mAB&#39;er </td><td> Biolegend </td><td> 103116 </td><td> Kan anvendes med forskellige fluoroforer (f.eks APC) </td></tr><tr><td> Paraformaldehyde (16%) </td><td> EMS Inc </td><td> 15710 </td><td> Fortynd 1:15 i 1X PBS </td></tr><tr><td> 15% TBE-urea-gel </td><td> Life Technologies Inc. </td><td> EC68855BOX </td><td></td></tr><tr><td> TaqMan MicroRNA Reverse Transcription Kit </td><td> Life Technologies Inc </td><td> 4366596 </td><td></td></tr><tr><td> TaqMan Universal PCR Master Mix </td><td> Life Technologies Inc </td><td> 4304437 </td><td></td></tr><tr><td> TaqMan MicroRNA Analyser mmu-miR-124a </td><td> Life Technologies Inc </td><td> 4427975 id 001182 </td><td></td></tr><tr><td> TaqMan MicroRNA Assays snoRNA-55 </td><td> Life Technologies Inc </td><td> 4427975 id 001228 </td><td></td></tr><tr><td> Nuklease-frit vand </td><td> Life Technologies Inc </td><td> AM9937 </td><td> Nuklease-frit vand </td></tr><tr><td> 384-godt klar optisk reaktionsplade </td&gt; <td> Life Technologies Inc. </td><td> 4309849 </td><td> Kan være substitueret med andre plader (f.eks plade med 96 brønde) i forskellige real-time PCR instrumenter </td></tr><tr><td> Dounce-homogenisator (15 ml) </td><td> Wheaton Science produkter. </td><td> 358044 </td><td> Teflon / glas </td></tr><tr><td> 40 μ nylon cellefilter </td><td> Falcon </td><td> 352340 </td><td></td></tr><tr><td> Big centrifuge </td><td> Sorval </td><td> RT 6000B </td><td> Rotor diameter 18,5 cm </td></tr><tr><td> Lille 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> Kan være substitueret med andre instrumenter </td></tr><tr><td> Flowcytometer </td><td> BD Biosciencess </td><td> LSR II </td><td> Kan være substitueret med andre instrumenter </td&gt; </tr><tr><td> FACS </td><td> BD Biosciences </td><td> FACSAria </td><td> Kan være substitueret med andre instrumenter </td></tr><tr><td> Nanodrop spektrofotometer </td><td> Thermo Scientific </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

Mikroglia er celler af myeloid herkomst, som ligger i det centrale nervesystem (CNS) 1. Disse celler spiller en vigtig rolle i sygdomme i mange sygdomme forbundet med neuroinflammation såsom dissemineret sklerose (MS) 2. Mikroglia i en normal CNS udtrykke makrofag markør CD11b og udviser en hvilende fænotype ved at udtrykke lave niveauer af aktivering markører såsom CD45. Under patologiske begivenheder i centralnervesystemet, er blevet microglia aktiveres som bestemt ved opregulering af CD45 og andre markører 3. De faktorer, der påvirker microglia fænotype og funktion i CNS er ikke godt undersøgt. MicroRNA (miRNA) er en voksende familie af konserverede molekyler (ca. 22 nukleotider lange), der er involveret i mange normale fysiologiske processer, såsom cellevækst og-differentiering 4 og patologier, såsom inflammation 5. MiRNA nedregulere ekspressionen af ​​visse målgener ved at binde komplementære sekvenserir mRNA&#39;er og spiller en vigtig rolle i aktiveringen af medfødte immunceller, herunder makrofager 6 og mikroglia 7. For at undersøge miRNA-medierede veje, som definerer mikroglial fænotype, biologisk funktion, og til at skelne microglia fra andre typer af makrofager, er det vigtigt at kvantitativt at vurdere ekspressionen af ​​bestemte microRNA&#39;er i særskilte undergrupper af CNS-resident microglia. Almindelige metoder til måling af ekspression af miRNA i CNS omfatter kvantitativ PCR fra hele neuronvæv og in situ hybridisering. Imidlertid ikke kvantitativ PCR fra hele vævshomogenat ikke tillade vurdering af ekspressionen af ​​miRNA i microglia, som kun udgør 5-15% af cellerne i neuronalt væv. Hybridisering in situ tillader vurdering af ekspressionen af microRNA i specifikke celletyper i vævssnit, men denne metode er ikke helt kvantitativ. I denne rapport beskriver vi en kvantitativ og sensitive fremgangsmåde til påvisning af miRNA ved real-time PCR microglia isoleret fra normale CNS eller under neuroinflammation hjælp eksperimentel autoimmun encephalomyelitis (EAE), en muse-model for MS. Den beskrevne fremgangsmåde vil være nyttig til at måle niveauet af ekspression af microRNA&#39;er i mikroglia i normale CNS eller under neuroinflammation forbundet med forskellige patologier, herunder MS, slagtilfælde, traumatisk skade, Alzheimers sygdom og hjernetumorer.

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

Mikroglia er bosiddende makrofager, der giver den første linje i forsvaret og immun-overvågning af det centrale nervesystem. MicroRNA er regulatoriske molekyler, som spiller en vigtig rolle i mange fysiologiske processer, herunder aktivering og differentiering af makrofager. I denne artikel beskriver vi metoden til måling af microRNA i mikroglia.

Påvisning af MicroRNA i Mikroglia af Real-time PCR i Normal CNS og under 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 ...

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Immunology and Infection

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

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