Name: polysome profiling in leishmania, human cells and mouse testis
Uploaded: 2018-04-08T15:00:00
Description: Watch this Scientific Journal Video about Polysome Profiling in Leishmania, Human Cells and Mouse Testis at JoVE.com

De auteurs bedanken Ching Lee voor hulp met audio-opname. Het onderzoek werd gesteund door de Start-up middelen uit Texas Tech University Health Sciences Center en door het Center of Excellence voor translationele neurowetenschappen en Therapeutics (CTNT) toekennen PN-CTNT 2017-05 AKHRJDHW A.L.K.; gedeeltelijk door de NIH grant R01AI099380 K.Z. James C. Huffman en Kristen R. Baca waren CISER (centrum voor de integratie van STEM onderwijs & onderzoek) geleerden en werden ondersteund door het programma.

Alle dierlijke behandelingen en hanteren van weefsels verkregen in de studie werden uitgevoerd volgens protocollen die zijn goedgekeurd door de institutionele Animal Care en gebruik Comité bij de Texas Tech University Health Science Center volgens de National Institutes of Gezondheid dierenwelzijn richtsnoeren, protocolnummer 96005. Gelieve te offeren van gewervelde dieren en weefsels volgens de richtlijnen van het institutionele Animal Care en gebruik Comité voor te bereiden. Als bij gebrek aan een dergelijk comité, ...

<ol>
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A., Lee, M. T., Giraldez, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ribosome+profiling+shows+that+miR-430+reduces+translation+before+causing+mRNA+decay+in+zebrafish.">Ribosome profiling shows that miR-430 reduces translation before causing mRNA decay in zebrafish.</a> Science. 336 (6078), 233-237 (2012).</li><li>Zanchin, N. I., Goldfarb, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nip7p+interacts+with+Nop8p,+an+essential+nucleolar+protein+required+for+60S+ribosome+biogenesis,+and+the+exosome+subunit+Rrp43p.">Nip7p interacts with Nop8p, an essential nucleolar protein required for 60S ribosome biogenesis, and the exosome subunit Rrp43p.</a> Molecular Cell Biology. 19 (2), 1518-1525 (1999).</li><li>Darnell, J. 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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Short+RNAs+repress+translation+after+initiation+in+mammalian+cells.">Short RNAs repress translation after initiation in mammalian cells.</a> Molecular Cell. 21 (4), 533-542 (2006).</li><li>Juntawong, P., Girke, T., Bazin, J., Bailey-Serres, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Translational+dynamics+revealed+by+genome-wide+profiling+of+ribosome+footprints+in+Arabidopsis.">Translational dynamics revealed by genome-wide profiling of ribosome footprints in Arabidopsis.</a> Proceedings of the National Academy of Science, USA. 111 (1), E203-E212 (2014).</li><li>Bunnik, E. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polysome+profiling+reveals+translational+control+of+gene+expression+in+the+human+malaria+parasite+Plasmodium+falciparum.">Polysome profiling reveals translational control of gene expression in the human malaria parasite Plasmodium falciparum.</a> Genome Biology. 14 (11), R128 (2013).</li><li>De Gaudenzi, J. G., Noe, G., Campo, V. A., Frasch, A. C., Cassola, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+expression+regulation+in+trypanosomatids.">Gene expression regulation in trypanosomatids.</a> Essays in Biochemistry. 51, 31-46 (2011).</li><li>Alves, L. R., Goldenberg, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA-binding+proteins+related+to+stress+response+and+differentiation+in+protozoa.">RNA-binding proteins related to stress response and differentiation in protozoa.</a> World Journal of Biological Chemistry. 7 (1), 78-87 (2016).</li><li>De Pablos, L. M., Ferreira, T. R., Walrad, P. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+differentiation+in+Leishmania+lifecycle+progression:+post-transcriptional+control+conducts+the+orchestra.">Developmental differentiation in Leishmania lifecycle progression: post-transcriptional control conducts the orchestra.</a> Current Opinions in Microbiology. 34, 82-89 (2016).</li><li>Soto, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-cycle-dependent+translation+of+histone+mRNAs+is+the+key+control+point+for+regulation+of+histone+biosynthesis+in+Leishmania+infantum.">Cell-cycle-dependent translation of histone mRNAs is the key control point for regulation of histone biosynthesis in Leishmania infantum.</a> Biochemical Journal. 379, 617-625 (2004).</li><li>McNicoll, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+3+'-untranslated+region+elements+regulate+stage-specific+mRNA+accumulation+and+translation+in+Leishmania.">Distinct 3 '-untranslated region elements regulate stage-specific mRNA accumulation and translation in Leishmania.</a> Journal of Biological Chemistry. 280 (42), 35238-35246 (2005).</li><li>Folgueira, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+translational+efficiencies+of+the+two+Leishmania+infantum+HSP70+mRNAs,+differing+in+their+3+'-untranslated+regions,+are+affected+by+shifts+in+the+temperature+of+growth+through+different+mechanisms.">The translational efficiencies of the two Leishmania infantum HSP70 mRNAs, differing in their 3 '-untranslated regions, are affected by shifts in the temperature of growth through different mechanisms.</a> Journal of Biological Chemistry. 280 (42), 35172-35183 (2005).</li><li>Dumas, C., Chow, C., Muller, M., Papadopoulou, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+class+of+developmentally+regulated+noncoding+RNAs+in+Leishmania.">A novel class of developmentally regulated noncoding RNAs in Leishmania.</a> Eukaryotic Cell. 5 (12), 2033-2046 (2006).</li><li>Kapler, G. 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L., Gorospe, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polysome+Fractionation+to+Analyze+mRNA+Distribution+Profiles.">Polysome Fractionation to Analyze mRNA Distribution Profiles.</a> Bio Protocols. 7 (3), (2017).</li><li>Sambrook, J., Fritsch, E. F., Maniatis, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular Cloning. A Laboratory Manual. , (1989).</li><li>Patrick, A. E., Karamyshev, A. L., Millen, L., Thomas, P. 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Polysoom fractionering door sacharose verloop in combinatie met RNA en eiwit analyse van breuken is een krachtige methode voor het analyseren van translationeel status van individuele mRNAs of de hele translatome en rollen van proteïne factoren reguleren translationeel machine tijdens normale fysiologische of ziekte staat. Polysomal profilering is een speciaal geschikt techniek te studeren translationeel verordening in organismen zoals trypanosomatids, met inbegrip van Leishmania waar Transcriptionele controle ...

Verordening van genexpressie in cellen wordt gecontroleerd door middel van de mechanismen van transcriptionele, posttranscriptional en posttranslationele. Vooruitgang in de diepe RNA sequencing toestaan de studie van de stationaire toestand mRNA niveaus op de schaal van een genoom-brede op een ongeëvenaard niveau. Echter, recente bevindingen bleek dat steady-state mRNA niveau niet altijd met eiwit productie1,2 correleren. Het lot van een individuele transcript is zeer complex en hangt af van vele factoren zoals interne/externe prikkels, stress, enz. Verordening van de genexpressie tijdens de eiwitsynthese biedt een andere laag van expressie controle nodig zijn voor een snelle reactie in veranderende omstandigheden. Polysoom (of "polyribosome") profilering, de scheiding en de visualisatie van actief vertalen ribosomen, is een krachtige methode om te studeren van de regulering van de eiwitsynthese. Hoewel, de eerste experimentele toepassingen verscheen in de jaren 1960-3, is Polysoom profilering momenteel een van de belangrijkste technieken in eiwit vertaling studies4. Enkele mRNAs kan worden vertaald door meer dan één ribosoom leidt tot de vorming van een Polysoom. Afschriften kunnen worden geblokkeerd op ribosomen met cycloheximide5 en mRNAs met verschillende nummers van polysomes kunnen worden gescheiden in het proces van Polysoom fractionering door sacharose kleurovergang ultracentrifugatie6,7 , 8 , 9. RNA analyse van polysomal breuken laat dan meting van veranderingen in de translationeel Staten van individuele mRNAs op genoom-brede schaal en tijdens verschillende fysiologische omstandigheden4,7, 10. de methode is ook gebruikt voor het onthullen van de rollen van 5' UTR en 3' UTR sequenties in controle van mRNA vertaalbaarheid11, de rol van miRNAs in translationeel repressie12onderzoeken, ontdekken van defecten in ribosoom biogenese13 , en begrijpen van de rol van ribosoom-geassocieerde eiwitten met ziekten bij de mens14,15. Tijdens de afgelopen tien jaar, is een steeds grotere rol voor de regulering van de genexpressie tijdens de vertaling gebleken dat het belang ervan voor ziekten bij de mens illustreert. Het bewijs voor translationeel controle in kanker, metabole en neurodegeneratieve ziekten is overweldigend15,16,17,18. Bijvoorbeeld disregulatie van eIF4E-afhankelijke vertalende controle draagt bij aan autisme gerelateerde tekorten15 en FMRP is betrokken bij vastlopen van ribosomen op mRNAs gekoppeld aan autisme14. Polysomal profilering is dus een zeer belangrijk instrument om te studeren fouten in translationeel verordening in meerdere ziekten bij de mens.
Eiwit analyse van polysomal breuken onder verschillende fysiologische omstandigheden ontleedt de functie van factoren die samenhangen met ribosomen tijdens de vertaling. De Polysoom profiling techniek is gebruikt in vele soorten, met inbegrip van gist, cellen van zoogdieren, planten en protozoa10,19,20,21. Eencellige parasieten zoals Trypanosoma en Leishmania vertonen beperkte Transcriptionele controle op de genexpressie. Hun genoom worden ingedeeld in polycistronic gen clusters die geen promotor-gereglementeerde transcriptie22. In plaats daarvan, developmental genexpressie wordt voornamelijk geregeld op het niveau van eiwit vertaling en mRNA stabiliteit in trypanosomatid soorten23,24. Begrip voor translationeel control in de afwezigheid van transcriptionele verordening is daarom bijzonder belangrijk voor deze organismen. Polysomal profilering is een krachtig hulpmiddel om te studeren van posttranscriptional regulering van genexpressie in Leishmania25,26,27,28.
De recente vooruitgang in de opsporing van individuele mRNAs niveaus door real time kwantitatieve PCR (RT-qPCR) en volledige transcriptome door next-generation sequencing, evenals proteomics technologieën, resolutie en voordelen van polysomal profielen naar een nieuw niveau brengt. Het gebruik van deze methoden kan worden uitgebreid door analyse van individuele polysomal breuken door diepe RNA sequencing gecombineerd met Proteoom analyse om de translationeel status van cellen in een genoom-brede schaal te controleren. Hierdoor is de identificatie van nieuwe moleculaire spelers regulering van de vertaling in de verschillende fysiologische en pathologische omstandigheden. Hier presenteren we een universele Polysoom profilering protocol dat wordt gebruikt op drie verschillende modellen: de parasiet, Leishmania grote, gekweekte menselijke cellen en dierlijke weefsels. Over de voorbereiding van cel lysates presenteren we advies van verschillende organismen, optimalisatie van de helling, mits het, keuze van RNase remmers en toepassing van RT-qPCR, westelijke vlek en RNA elektroforese te analyseren Polysoom breuken in deze studie.

<table>
	<tbody>
		<tr>
			<td>Instruments:</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Gradient master</td>
			<td>Biocomp Instruments Inc.</td>
			<td>108</td>
		</tr>
		<tr>
			<td>Piston Gradient Fractionator</td>
			<td>Biocomp Instruments Inc.</td>
			<td>152</td>
		</tr>
		<tr>
			<td>Fraction collector</td>
			<td>Gilson, Inc.</td>
			<td>FC203B</td>
		</tr>
		<tr>
			<td>NanoDrop One</td>
			<td>Thermo Scientific</td>
			<td>NanoDrop One</td>
		</tr>
		<tr>
			<td>Nikon inverted microscope</td>
			<td>Nikon</td>
			<td>ECLIPSE Ts2-FL/Ts2</td>
		</tr>
		<tr>
			<td>2720 Thermal Cycler</td>
			<td>Applied Biosystems by Life Technologies</td>
			<td>4359659</td>
		</tr>
		<tr>
			<td>CO2 incubator</td>
			<td>Panasonic Healthcare Co.</td>
			<td>MCO-170A1CUV</td>
		</tr>
		<tr>
			<td>HERATHERM incubator</td>
			<td>Thermo Scientific</td>
			<td>51028063</td>
		</tr>
		<tr>
			<td>Biological Safety Cabinet, class II, type A2</td>
			<td>NuAire Inc.</td>
			<td>NU-543-400</td>
		</tr>
		<tr>
			<td>Revco freezer</td>
			<td>Revco Technologies</td>
			<td>ULT1386-5-D35</td>
		</tr>
		<tr>
			<td>Beckman L8-M Ultracentifuge</td>
			<td>Beckman Coulter</td>
			<td>L8M-70</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf</td>
			<td>5810R</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf</td>
			<td>5424</td>
		</tr>
		<tr>
			<td>Ultracentrifuge Rotor SW41</td>
			<td>Beckman Coulter</td>
			<td>331362</td>
		</tr>
		<tr>
			<td>Swing-bucket rotor</td>
			<td>Eppendorf</td>
			<td>A-4-62</td>
		</tr>
		<tr>
			<td>Fixed angle rotor</td>
			<td>Eppendorf</td>
			<td>F-45-30-11</td>
		</tr>
		<tr>
			<td>Quant Studio 12K Flex Real-Time PCR machine 285880228</td>
			<td>Applied Biosystems by life technologies</td>
			<td>4470661</td>
		</tr>
		<tr>
			<td>TC20 Automated cell counter</td>
			<td>Bio-Rad</td>
			<td>145-0102</td>
		</tr>
		<tr>
			<td>Hemacytometer</td>
			<td>Hausser Scientific</td>
			<td>02-671-51B</td>
		</tr>
		<tr>
			<td>Software&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Triax software&#160;</td>
			<td>Biocomp Instruments Inc.</td>
			<td></td>
		</tr>
		<tr>
			<td>Materials:</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Counting slides, dual chamber for cell counter</td>
			<td>Bio-Rad</td>
			<td>145-0011</td>
		</tr>
		<tr>
			<td>1.5 mL microcentrifuge tube</td>
			<td>USA Scientific</td>
			<td>1615-5500</td>
		</tr>
		<tr>
			<td>Open-top polyclear centrifuge tubes, (14 mm x 89 mm)</td>
			<td>Seton Scientific</td>
			<td>7030</td>
		</tr>
		<tr>
			<td>Syringe, 5 mL</td>
			<td>BD</td>
			<td>309646</td>
		</tr>
		<tr>
			<td>BD Syringe 3 mL23 Gauge 1 Inch Needle</td>
			<td>BD</td>
			<td>10020439</td>
		</tr>
		<tr>
			<td>Nunclon Delta Surface plate, 14 cm</td>
			<td>Thermo Scientific</td>
			<td>168381</td>
		</tr>
		<tr>
			<td>Nunclon Delta Surface plate, 9 cm</td>
			<td>Thermo Scientific</td>
			<td>172931</td>
		</tr>
		<tr>
			<td>Nalgene rapid-flow 90mm filter unit, 500 mL, 0.2 aPES</td>
			<td>Thermo Scientific</td>
			<td>569-0020</td>
		</tr>
		<tr>
			<td>BioLite 75 cm3 flasks</td>
			<td>Thermo Scientific</td>
			<td>130193</td>
		</tr>
		<tr>
			<td>Nunc 50 mL conical centrifuge tubes</td>
			<td>Thermo Scientific</td>
			<td>339653</td>
		</tr>
		<tr>
			<td>Chemicals:</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Trizol LS</td>
			<td>Ambion by Life Technologies</td>
			<td>10296028</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Fisher Scientific</td>
			<td>BP310-500</td>
		</tr>
		<tr>
			<td>Trizma base</td>
			<td>Sigma</td>
			<td>T1378-5KG</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle&#39;s Medium-high glucose (DMEM)</td>
			<td>Sigma</td>
			<td>D6429-500ML</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Sigma</td>
			<td>F0926-50ML</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin (P/S)</td>
			<td>Sigma</td>
			<td>P0781-100ML</td>
		</tr>
		<tr>
			<td>Lipofectamine 2000</td>
			<td>Invitrogen</td>
			<td>11668-019</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s phosphate buffered saline (DPBS)</td>
			<td>Sigma</td>
			<td>D8537-500ML</td>
		</tr>
		<tr>
			<td>Magnesium chloride hexahydrate (MgCl2x6H2O)</td>
			<td>Acros Organics</td>
			<td>AC413415000</td>
		</tr>
		<tr>
			<td>Potassium Chloride (KCl)</td>
			<td>Sigma</td>
			<td>P9541-500G</td>
		</tr>
		<tr>
			<td>Nonidet P 40 (NP-40)</td>
			<td>Fluka (Sigma-Aldrich)</td>
			<td>74385</td>
		</tr>
		<tr>
			<td>Recombinant Rnasin Ribonuclease Inhibitor</td>
			<td>Promega</td>
			<td>N2511</td>
		</tr>
		<tr>
			<td>Heparin sodium salt</td>
			<td>Sigma</td>
			<td>H3993-1MU</td>
		</tr>
		<tr>
			<td>cOmplete Mini EDTA-free protease inhibitors</td>
			<td>Roche Diagnostics</td>
			<td>11836170001</td>
		</tr>
		<tr>
			<td>Glycogen</td>
			<td>Thermo Scientific</td>
			<td>R0551</td>
		</tr>
		<tr>
			<td>Water</td>
			<td>Sigma</td>
			<td>W4502-1L</td>
		</tr>
		<tr>
			<td>Cycloheximide</td>
			<td>Sigma</td>
			<td>C7698-1G</td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Fisher Scientific</td>
			<td>194002</td>
		</tr>
		<tr>
			<td>Dithiotreitol (DTT)</td>
			<td>Fisher Scientific</td>
			<td>BP172-5</td>
		</tr>
		<tr>
			<td>Ethidium Bromide</td>
			<td>Fisher Scientific</td>
			<td>BP-1302-10</td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid disodium dehydrate (EDTA)</td>
			<td>Fisher Scientific</td>
			<td>S316-212</td>
		</tr>
		<tr>
			<td>Optimem</td>
			<td>Life Technologies</td>
			<td>22600050</td>
		</tr>
		<tr>
			<td>Puromycin dihydrochloride</td>
			<td>Sigma</td>
			<td>P8833-100MG</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Fisher Scientific</td>
			<td>S5-3KG</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA solution</td>
			<td>Sigma</td>
			<td>T4049-100ML</td>
		</tr>
		<tr>
			<td>Hgh Capacity cDNA Reverse Transcriptase Kit</td>
			<td>Applied Biosystems by life technologies</td>
			<td>4368814</td>
		</tr>
		<tr>
			<td>Power SYBR Green PCR Master Mix</td>
			<td>Applied Biosystems by life technologies</td>
			<td>4367659</td>
		</tr>
		<tr>
			<td>HCl</td>
			<td>Fisher Scientific</td>
			<td>A144SI-212</td>
		</tr>
		<tr>
			<td>Isopropanol</td>
			<td>Fisher Scientific</td>
			<td>BP26324</td>
		</tr>
		<tr>
			<td>Potassium Hydroxide (KOH)</td>
			<td>Sigma</td>
			<td>221473-500G</td>
		</tr>
		<tr>
			<td>Anti-RPL11 antibody</td>
			<td>Abcam</td>
			<td>ab79352</td>
		</tr>
		<tr>
			<td>Ribosomal protein S6 (C-8) antibody</td>
			<td>Santa Cruz Biotechnology Inc.</td>
			<td>sc-74459</td>
		</tr>
		<tr>
			<td>1xM199</td>
			<td>Sigma</td>
			<td>M0393-10X1L</td>
		</tr>
		<tr>
			<td>Lithium cloride</td>
			<td>Sigma</td>
			<td>L-9650</td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO)</td>
			<td>Fisher Scientific</td>
			<td>D128-500</td>
		</tr>
		<tr>
			<td>Gel Loading Buffer II</td>
			<td>Thermo Scientific</td>
			<td>AM8546G</td>
		</tr>
		<tr>
			<td>UltraPure Agarose</td>
			<td>Thermo Scientific</td>
			<td>16500-100</td>
		</tr>
		<tr>
			<td>Trichloracetic acid (TCA)</td>
			<td>Fisher Scientific</td>
			<td>A322-100</td>
		</tr>
		<tr>
			<td>SuperSignal West Pico PLUS chemiluminescent substrate</td>
			<td>Thermo Scientific</td>
			<td>34580</td>
		</tr>
		<tr>
			<td>Formaldehyde</td>
			<td>Fisher Scientific</td>
			<td>BP531-500</td>
		</tr>
		<tr>
			<td>Sodium Dodecyl Sulfate (SDS)</td>
			<td>Sigma</td>
			<td>L5750-1KG</td>
		</tr>
		<tr>
			<td>Phenylmethylsulfonyl fluoride (PMSF)</td>
			<td>Sigma</td>
			<td>P7626-5G</td>
		</tr>
		<tr>
			<td>RNeasy Mini kit</td>
			<td>Qiagen</td>
			<td>74104</td>
		</tr>
		<tr>
			<td>Adenosine 5&#8242;-triphosphate disodium salt hydrate (ATP)</td>
			<td>Sigma</td>
			<td>A1852-1VL</td>
		</tr>
		<tr>
			<td>Cytosine 5&#39;-triphosphate disodium salt hydrate (CTP)</td>
			<td>Sigma</td>
			<td>C1506-250MG</td>
		</tr>
		<tr>
			<td>Uridine 5&#39;-triphosphate trisodium salt hydrate (UTP)</td>
			<td>Sigma</td>
			<td>U6625-100MG</td>
		</tr>
		<tr>
			<td>Guanosine 5&#39;-triphosphate sodium salt hydrate (GTP)</td>
			<td>Sigma</td>
			<td>G8877-250MG</td>
		</tr>
		<tr>
			<td>SP6 RNA Polymerase</td>
			<td>NEB</td>
			<td>M0207S</td>
		</tr>
		<tr>
			<td>Pyrophoshatase</td>
			<td>Sigma</td>
			<td>I1643-500UN</td>
		</tr>
		<tr>
			<td>Spermidine</td>
			<td>Sigma</td>
			<td>S0266-1G</td>
		</tr>
	</tbody>
</table>

polysome profiling in leishmania, human cells and mouse testis

Goede eiwit expressie op het juiste moment en in de juiste hoeveelheden is de basis van normale cel functie en overleven in een snel veranderende omgeving. Voor een lange tijd, werden de gen expressie studies gedomineerd door onderzoek op het transcriptional niveau. Echter de steady-state niveaus van mRNAs doen niet correleren goed met de productie van eiwitten en de vertaalbaarheid van mRNAs varieert afhankelijk van de omstandigheden. In sommige organismen, zoals de parasiet, Leishmania, is het eiwit expressie meestal op de translationeel niveau geregeld. Recente studies aangetoond dat eiwitten vertaling disregulatie wordt geassocieerd met kanker, metabole, neurodegeneratieve en andere ziekten bij de mens. Polysoom profilering is een krachtige methode om te studeren eiwit vertaling verordening. U kunt de translationeel status van individuele mRNAs meten of de vertaling op de schaal van een genoom-brede onderzoeken. De basis van deze techniek is de scheiding van polysomes, ribosomen, subeenheden en gratis mRNAs tijdens centrifugeren van een cytoplasmatische lysate via een verloop van sacharose. Hier presenteren we een universele Polysoom profiling protocol dat wordt gebruikt op drie verschillende modellen - parasiet, Leishmania grote, gekweekte menselijke cellen en dierlijke weefsels. Leishmania cellen groeien vrij in suspensie en gekweekte menselijke cellen groeien in aanhangend enkelgelaagde, terwijl muis testis een dierlijk weefsel steekproef. De techniek is dus aangepast aan elk van deze bronnen. Het protocol voor de analyse van polysomal breuken bevat detectie van individuele mRNA niveaus door RT-qPCR, eiwitten door westelijke vlek en analyse van ribosomaal RNAs door elektroforese. De methode kan worden uitgebreid door onderzoek van mRNAs vereniging met het ribosoom op een niveau van transcriptome door diepe RNA-seq en analyse van ribosoom-geassocieerde eiwitten door massaspectrometrie van de fracties. De methode kan gemakkelijk worden aangepast aan andere biologische modellen.

In deze studie, beschrijven we de toepassing van de polysomal profiling techniek op drie verschillende bronnen: parasitaire Leishmania grote, gekweekte menselijke cellen en muis testis. Leishmania cellen groeien vrij in de vloeibare media in suspensie, gekweekte menselijke cellen groeien in het aanhangend enkelgelaagde op platen, en de testis muis een weefsel steekproef. De methode kan gemakkelijk worden aangepast aan andere soorten vrij volwassen cellen in schorsing, ve...

Watch this Scientific Journal Video about Polysome Profiling in Leishmania, Human Cells and Mouse Testis at JoVE.com

Polysome Profiling in Leishmania, Human Cells and Mouse Testis

Het algemene doel van Polysoom profiling techniek is analyse van translationeel activiteiten van individuele mRNAs of transcriptome mRNAs tijdens de eiwitsynthese. De methode is belangrijk voor de studie van eiwit synthese verordening, vertaling activering en onderdrukking in gezondheid en meerdere ziekten bij de mens.

Polysoom profilering in Leishmania, menselijke cellen en muis Testis

Proper protein expression at the right time and in the right amounts is the basis of normal cell function and survival in a fast-changing environment. For ...

Research

JoVE Journal

Biochemistry

Polysome Profiling in Leishmania, Human Cells and Mouse Testis

Thermostabilization, Expression, Purification, and Crystallization of the Human Serotonin Transporter Bound to S-citalopram

Thermostabilization, Expression, Purification, and Crystallization of the Human Serotonin Transporter Bound to S-citalopram

Thermostabilization, expressie, zuivering en kristallisatie van het Human serotonine transporter Gebonden aan<em&gt; S</em&gt; citalopram

The serotonin transporter is a sodium and chloride-coupled transporter that &#34;pumps&#34; extracellular serotonin into cells. S-citalopram is a drug ...

Genome-wide Quantification of Translation in Budding Yeast by Ribosome Profiling

Genoom-brede kwantificering van vertaling in de ontluikende gist door het ribosoom profilering

Translation of mRNA into proteins is a complex process involving several layers of regulation. It is often assumed that changes in mRNA transcription ...

Calibration-free In Vitro Quantification of Protein Homo-oligomerization Using Commercial Instrumentation and Free, Open Source Brightness Analysis Software

Calibration-free In Vitro Quantification of Protein Homo-oligomerization Using Commercial Instrumentation and Free, Open Source Brightness Analysis Software

Kalibratie-gratis In Vitro kwantificering van eiwit Homo-oligomerisatie met behulp van commerciële instrumentatie en Software van de analyse van de helderheid van gratis, Open Source

Number and brightness is a calibration-free fluorescence fluctuation spectroscopy (FFS) technique for detecting protein homo-oligomerization. It can be ...

Dissipative Microgravimetry to Study the Binding Dynamics of the Phospholipid Binding Protein Annexin A2 to Solid-supported Lipid Bilayers Using a Quartz Resonator

Dissipatief Microgravimetry te bestuderen van de dynamiek van de Binding van de fosfolipide bindend proteïne Annexine A2 aan lipide Solid-ondersteund Bilayers met behulp van een Resonator Quartz

The dissipative quartz crystal microbalance technique is a simple and label-free approach to measure simultaneously the mass uptake and viscoelastic ...

Measuring and Interpreting Oxygen Consumption Rates in Whole Fly Head Segments

Meten en interpreteren van zuurstof verbruik geheel vliegen hoofd segmenten

Regulated metabolic activity is essential for the normal functioning of living cells. Indeed, altered metabolic activity is causally linked with the ...

Nitropeptide Profiling and Identification Illustrated by Angiotensin II

Nitropeptide profilering en identificatie geïllustreerd door angiotensine II

Protein nitration is one of the most important post-translational modifications (PTM) on tyrosine residues and it can be induced by chemical actions of ...

High-Resolution Complexome Profiling by Cryoslicing BN-MS Analysis

Hoge-resolutie Complexome profilering door Cryoslicing BN-MS analyse

Proteins generally exert biological functions through interactions with other proteins, either in dynamic protein assemblies or as a part of stably formed ...

Profiling Ubiquitin and Ubiquitin-like Dependent Post-translational Modifications and Identification of Significant Alterations

Profilering van Ubiquitin en Ubiquitin-achtige afhankelijke post-translationele modificaties en identificatie van belangrijke veranderingen

Ubiquitin (ub) and ubiquitin-like (ubl) dependent post-translational modifications of proteins play fundamental biological regulatory roles within the ...

Phosphoproteomic Strategy for Profiling Osmotic Stress Signaling in Arabidopsis

Phosphoproteomic Strategy for Profiling Osmotic Stress Signaling in Arabidopsis

Fosfaatprotemische strategie voor profilering osmotische stresssignalering in Arabidopsis

Protein phosphorylation is crucial for the regulation of enzyme activity and gene expression under osmotic condition. Mass spectrometry (MS)-based ...

Ganglioside Extraction, Purification and Profiling

Ganglioside extractie, zuivering en profilering

Gangliosides are glycosphingolipids that contain one or more sialic acid residues. They are found on all vertebrate cells and tissues but are especially ...