Name: dynamic proteomic and mirna analysis of polysomes from isolated mouse heart after langendorff perfusion
Uploaded: 2018-08-29T13:00:00
Description: Watch this Scientific Journal Video about Dynamic Proteomic and miRNA Analysis of Polysomes from Isolated Mouse Heart After Langendorff Perfusion at JoVE.com

HL112730 NIH P01 (pano, JVE), NIH R01 HL132075 (pano, JVE), centro do coração feminino-Barbra Streisand (pano, JVE), Dorothy e Lyon de Phillip E. cadeira em Cardiologia Molecular (RAG), Erika Glazer dotado cadeira na saúde do coração feminino (JVE) e a Academia de Ciências da República Checa Apoio institucional RVO: 68081715 (MS).

Todos os estudos em animais foram realizados em conformidade com as orientações institucionais e aprovados pelo Comitê de uso do Cedars-Sinai Medical Center e institucional Cuidado Animal.
1. Langendorff perfusão do coração de rato
<ol><li>
Perfusão de Langendorff de coração de rato com isquemia e reperfusão
	<ol>
<li>Administrar intraperitoneal de pentobarbital de sódio 70 mg/kg para o rato adulto (8 semanas, macho, C57BL6/j). Confirme a anestesia profunda por fa...

<ol>
	<li>Pourpirali, S., Valacca, C., Merlo, P., Rizza, S., D&#8217;Amico, S., Cecconi, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prolonged+pseudohypoxia+targets+ambra1+mrna+to+p-bodies+for+translational+repression.">Prolonged pseudohypoxia targets ambra1 mrna to p-bodies for translational repression.</a> PLoS ONE. 10, e0129750 (2015).</li><li>Andres, A. M., Tucker, K. C., Thomas, A., Taylor, D. J., Sengstock, D., Jahania, S. M., Dabir, R., Pourpirali, S., Brown, J. A., Westbrook, D. G., Ballinger, S. W., Mentzer, R. M., Gottlieb, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitophagy+and+mitochondrial+biogenesis+in+atrial+tissue+of+patients+undergoing+heart+surgery+with+cardiopulmonary+bypass.">Mitophagy and mitochondrial biogenesis in atrial tissue of patients undergoing heart surgery with cardiopulmonary bypass.</a> JCI Insight. 2, e89303 (2017).</li><li>Kislinger, T., Cox, B., Kannan, A., Chung, C., Hu, P., Ignatchenko, A., Scott, M. S., Gramolini, A. O., Morris, Q., Hallett, M. T., Rossant, J., Hughes, T. R., Frey, B., Emili, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+survey+of+organ+and+organelle+protein+expression+in+mouse:+Combined+proteomic+and+transcriptomic+profiling.">Global survey of organ and organelle protein expression in mouse: Combined proteomic and transcriptomic profiling.</a> Cell. 125, 173-186 (2006).</li><li>Kren, B. T., Wong, P. Y., Shiota, A., Zhang, X., Zeng, Y., Steer, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polysome+trafficking+of+transcripts+and+micrornas+in+regenerating+liver+after+partial+hepatectomy.">Polysome trafficking of transcripts and micrornas in regenerating liver after partial hepatectomy.</a> American Journal of Physiology. Gastrointestinal and Liver Physiology. 297, G1181-G1192 (2009).</li><li>Gottlieb, R. A., Pourpirali, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lost+in+translation:+Mirnas+and+mrnas+in+ischemic+preconditioning+and+ischemia/reperfusion+injury.">Lost in translation: Mirnas and mrnas in ischemic preconditioning and ischemia/reperfusion injury.</a> Journal of Molecular and Cellular Cardiology. 95, 70-77 (2016).</li><li>Coudert, L., Adjibade, P., Mazroui, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+translation+initiation+during+stress+conditions+by+polysome+profiling.">Analysis of translation initiation during stress conditions by polysome profiling.</a> Journal of Visualized Experiments. , (2014).</li><li>Lorsch, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+in+enzymology.+Laboratory+methods+in+enzymology:+Rna.+Preface.">Methods in enzymology. Laboratory methods in enzymology: Rna. Preface.</a> Methods in Enzymology. 530, xxi (2013).</li><li>Kuzniewska, B., Chojnacka, M., Milek, J., Dziembowska, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+polysomal+fractions+from+mouse+brain+synaptoneurosomes+and+analysis+of+polysomal-bound+mrnas.">Preparation of polysomal fractions from mouse brain synaptoneurosomes and analysis of polysomal-bound mrnas.</a> Journal of Neuroscience Methods. 293, 226-233 (2018).</li><li>He, S. L., Green, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polysome+analysis+of+mammalian+cells.">Polysome analysis of mammalian cells.</a> Methods in Enzymology. 530, 183-192 (2013).</li><li>Molotski, N., Soen, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+association+of+micrornas+with+polysomes+reflects+distinct+strengths+of+interactions+with+their+mrna+targets.">Differential association of micrornas with polysomes reflects distinct strengths of interactions with their mrna targets.</a> RNA. 18, 1612-1623 (2012).</li><li>Maroney, P. A., Yu, Y., Fisher, J., Nilsen, T. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+that+micrornas+are+associated+with+translating+messenger+rnas+in+human+cells.">Evidence that micrornas are associated with translating messenger rnas in human cells.</a> Nature Structural and Molecular Biology. 13, 1102-1107 (2006).</li><li>Paul, P., Chakraborty, A., Sarkar, D., Langthasa, M., Rahman, M., Bari, M., Singha, R. S., Malakar, A. K., Chakraborty, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interplay+between+mirnas+and+human+diseases.">Interplay between mirnas and human diseases.</a> Journal of Cellular Physiology. 233, 2007-2018 (2018).</li><li>Rupaimoole, R., Slack, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microrna+therapeutics:+Towards+a+new+era+for+the+management+of+cancer+and+other+diseases.">Microrna therapeutics: Towards a new era for the management of cancer and other diseases.</a> Nature Reviews Drug Discovery. 16, 203-222 (2017).</li><li>Nelson, P. T., Hatzigeorgiou, A. G., Mourelatos, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mirnp:Mrna+association+in+polyribosomes+in+a+human+neuronal+cell+line.">Mirnp:Mrna association in polyribosomes in a human neuronal cell line.</a> RNA. 10, 387-394 (2004).</li><li>Nottrott, S., Simard, M. J., Richter, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+let-7a+mirna+blocks+protein+production+on+actively+translating+polyribosomes.">Human let-7a mirna blocks protein production on actively translating polyribosomes.</a> Nature Structural and Molecular Biology. 13, 1108-1114 (2006).</li><li>Kim, J., Krichevsky, A., Grad, Y., Hayes, G. D., Kosik, K. S., Church, G. M., Ruvkun, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+many+micrornas+that+copurify+with+polyribosomes+in+mammalian+neurons.">Identification of many micrornas that copurify with polyribosomes in mammalian neurons.</a> Proceedings of the National Academy of Sciences of the United States of America. , 360-365 (2004).</li><li>Androsavich, J. R., Chau, B. N., Bhat, B., Linsley, P. S., Walter, N. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disease-linked+microrna-21+exhibits+drastically+reduced+mrna+binding+and+silencing+activity+in+healthy+mouse+liver.">Disease-linked microrna-21 exhibits drastically reduced mrna binding and silencing activity in healthy mouse liver.</a> RNA. 18, 1510-1526 (2012).</li><li>Kraushar, M. L., Thompson, K., Wijeratne, H. R., Viljetic, B., Sakers, K., Marson, J. W., Kontoyiannis, D. L., Buyske, S., Hart, R. P., Rasin, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporally+defined+neocortical+translation+and+polysome+assembly+are+determined+by+the+rna-binding+protein+hu+antigen+r.">Temporally defined neocortical translation and polysome assembly are determined by the rna-binding protein hu antigen r.</a> Proceedings of the National Academy of Sciences of the United States of America. , E3815-E3824 (2014).</li><li>McClatchy, D. B., Ma, Y., Liu, C., Stein, B. D., Martinez-Bartolome, S., Vasquez, D., Hellberg, K., Shaw, R. J., Yates, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pulsed+azidohomoalanine+labeling+in+mammals+(palm)+detects+changes+in+liver-specific+lkb1+knockout+mice.">Pulsed azidohomoalanine labeling in mammals (palm) detects changes in liver-specific lkb1 knockout mice.</a> Journal of Proteome Research. 14, 4815-4822 (2015).</li><li>Schiapparelli, L. M., McClatchy, D. B., Liu, H. H., Sharma, P., Yates, J. R., Cline, H. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+detection+of+biotinylated+proteins+by+mass+spectrometry.">Direct detection of biotinylated proteins by mass spectrometry.</a> Journal of Proteome Research. 13, 3966-3978 (2014).</li><li>Wi&#347;niewski, J. R., Zougman, A., Nagaraj, N., Mann, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Universal+sample+preparation+method+for+proteome+analysis.">Universal sample preparation method for proteome analysis.</a> Nature Methods. 6, 359-362 (2009).</li><li>Rajalingam, D., Loftis, C., Xu, J. J., Kumar, T. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trichloroacetic+acid-induced+protein+precipitation+involves+the+reversible+association+of+a+stable+partially+structured+intermediate.">Trichloroacetic acid-induced protein precipitation involves the reversible association of a stable partially structured intermediate.</a> Protein Science. 18, 980-993 (2009).</li><li>Fic, E., Kedracka-Krok, S., Jankowska, U., Pirog, A., Dziedzicka-Wasylewska, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+protein+precipitation+methods+for+various+rat+brain+structures+prior+to+proteomic+analysis.">Comparison of protein precipitation methods for various rat brain structures prior to proteomic analysis.</a> Electrophoresis. 31, 3573-3579 (2010).</li><li>Sashital, D. G., Greeman, C. A., Lyumkis, D., Potter, C. S., Carragher, B., Williamson, J. 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+combined+quantitative+mass+spectrometry+and+electron+microscopy+analysis+of+ribosomal+30s+subunit+assembly+in+e.+Coli.">A combined quantitative mass spectrometry and electron microscopy analysis of ribosomal 30s subunit assembly in e. Coli.</a> Elife. 3, (2014).</li><li>Liang, S., Bellato, H. M., Lorent, J., Lupinacci, F. C. S., Oertlin, C., van Hoef, V., Andrade, V. P., Roff&#233;, M., Masvidal, L., Hajj, G. N. M., Larsson, O. <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+in+small+tissue+samples.">Polysome-profiling in small tissue samples.</a> Nucleic Acids Research. 46, e3 (2018).</li></ol>

Análise do perfil de polysome permite o estudo da tradução de proteínas, analisando o estado translacional de um mRNA específico ou o transcriptoma toda6,7. Também é de grande ajuda quando a tradução local precisa ser estudado como sinaptossomas8. Tradicionalmente, este método envolve a separação de mono e poliribosomami e os mRNAs associados em um gradiente de sacarose, que pode ser acoplado a genômica ou técnicas de proteô...

O coração responde para a lesão de reperfusão (R) de forma dinâmica e de isquemia (I). No entanto, há um pequeno insight agudas alterações na síntese de proteínas durante a resposta. Para resolver isso, aproveitamos o método bem estabelecido de polysome1 para identificar alterações na abundância de proteína que refletem a redistribuição dos ribossomas e translacionais fatores regulatórios do citosol para polissomos, de criação de perfil e o aumento de proteínas recém sintetizadas (PTS). Na configuração de I / R, o aumento da síntese de novas proteínas ocorre em um período de tempo que é inconsistente com a transcrição de mRNAs de novo2; Além disso, a discordância entre os níveis de expressão de mRNA e abundância de proteína tem sido relatados3. Por estas razões, nós escolhemos analisar as mudanças na proteome dinâmico como refletido pela tradução da proteína. Para fazer isso, podemos quantificar o mRNA nas fracções polysome e analisar a composição de proteína nas fracções polysome. Finalmente, porque microRNAs (miRs) regular a disponibilidade dos mRNAs para tradução e pode interferir com a eficácia da proteína tradução4,5, examinamos a distribuição de miRs nas fracções polysome, enfocando a resposta a I / r.
Optamos por usar o modelo de perfusão de Langendorff de rato isolado e tecidos colhidos em condições basais de perfusão contínua, após 30 min isquemia global de não-fluxo e após 30 min de isquemia seguida por 30 min de reperfusão. Em seguida, solubilizado no tecido do coração e separados polissomos ao longo de um gradiente de sacarose, seguido de análise proteômica e detecção seletiva de mRNAs e miRNAs por PCR e microarray, respectivamente. Esta combinação de métodos representa uma poderosa abordagem para a compreensão do proteome dinâmico, permitindo a detecção simultânea de mRNA, miRNA e pts, bem como a redistribuição de proteínas reguladoras, miRNA e mRNA entre frações nontranslating, polissomos de baixa eficiência e alta eficiência polissomos (ver Figura 1). Insights sobre o Regulamento dinâmico deste processo serão estendidas por uma análise mais aprofundada da fosforilação de fatores-chave reguladoras como eIF2α ou mTOR. Agora, estas etapas individuais são descritas em detalhes.

<table>
	<tbody>
		<tr>
			<td>Pentobarbital</td>
			<td>Vortech Phamaceuticals</td>
			<td>9373</td>
			<td>for euthansia</td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Sagent</td>
			<td>103424</td>
			<td>used in langendorff preparation</td>
		</tr>
		<tr>
			<td>forceps</td>
			<td>Fine Science Tools</td>
			<td>91110-10</td>
			<td>used to hang the heart</td>
		</tr>
		<tr>
			<td>Langendorff system</td>
			<td>Radnoti + home made</td>
			<td>n/a</td>
			<td>A &#39;four heart&#39; system consisting of custom blown glass, tubing and water baths</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma</td>
			<td>S7653-5KG</td>
			<td>Krebs buffer and Sucrose gradient</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma</td>
			<td>P5405</td>
			<td>Krebs buffer and Lysis buffer</td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>Sigma</td>
			<td>P-5504</td>
			<td>Krebs buffer</td>
		</tr>
		<tr>
			<td>MgSO4</td>
			<td>Sigma</td>
			<td>M7774-500G</td>
			<td>Krebs buffer</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma</td>
			<td>G5767</td>
			<td>Krebs buffer</td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Sigma</td>
			<td>C1016-500G</td>
			<td>Krebs buffer</td>
		</tr>
		<tr>
			<td>Sucrose powder</td>
			<td>Sigma</td>
			<td>S0389-1KG</td>
			<td>Sucrose gradient</td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Sigma</td>
			<td>208337</td>
			<td>Sucrose gradient and Lysis buffer</td>
		</tr>
		<tr>
			<td>Tris-base</td>
			<td>Sigma</td>
			<td>T1503-1KG</td>
			<td>Sucrose gradient and Lysis buffer</td>
		</tr>
		<tr>
			<td>Xylene Cyanole</td>
			<td>Sigma</td>
			<td>X-4126</td>
			<td>Sucrose gradient</td>
		</tr>
		<tr>
			<td>Cycloheximide</td>
			<td>Sigma-aldrich</td>
			<td>239763</td>
			<td>Sucrose gradient and Lysis buffer</td>
		</tr>
		<tr>
			<td>RNaseOUT</td>
			<td>Life Technologies</td>
			<td>C00019</td>
			<td>RNAse inhibitor for Lysis buffer</td>
		</tr>
		<tr>
			<td>Igepal CA-360 (NP40)</td>
			<td>Sigma</td>
			<td>I3021</td>
			<td>Lysis buffer</td>
		</tr>
		<tr>
			<td>Protease Inhibitor Cocktail tablets, EDTA free</td>
			<td>Roche</td>
			<td>5056489001</td>
			<td></td>
		</tr>
		<tr>
			<td>Tube, Thinwall, Ultra-Clear, 13.2 mL, 14 x 89 mm</td>
			<td>Beckman Coulter</td>
			<td>344059</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultracentrifuge</td>
			<td>Beckman</td>
			<td>LE-80K</td>
			<td>Ultracentrifugation of the gradients</td>
		</tr>
		<tr>
			<td>Rotor</td>
			<td>Beckman</td>
			<td>SW41</td>
			<td>Ultracentrifugation of the gradients</td>
		</tr>
		<tr>
			<td>Biologic LP (pump)</td>
			<td>Biorad</td>
			<td>731-8300</td>
			<td>Fractionation of the gradients</td>
		</tr>
		<tr>
			<td>BioFrac</td>
			<td>Biorad</td>
			<td>741-0002</td>
			<td>Fractionation of the gradients</td>
		</tr>
		<tr>
			<td>Eppendorf RNA/DNA LoBind microcentrifuge tubes, 2 mL tube</td>
			<td>Sigma</td>
			<td>Z666513-100EA</td>
			<td>Gradient fraction and RNA extraction</td>
		</tr>
		<tr>
			<td>TRIzol Reagent</td>
			<td>Life technologies</td>
			<td>AM9738</td>
			<td>RNA extraction</td>
		</tr>
		<tr>
			<td>Luciferase Control RNA</td>
			<td>Promega</td>
			<td>L4561</td>
			<td>RNA extraction</td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Fisher Scientific</td>
			<td>C606-4</td>
			<td>RNA extraction</td>
		</tr>
		<tr>
			<td>Glycogen, RNA grade</td>
			<td>Thermo Fisher Scientific</td>
			<td>R0551</td>
			<td>RNA extraction</td>
		</tr>
		<tr>
			<td>Isopropanol</td>
			<td>Sigma</td>
			<td>I9516</td>
			<td>RNA extraction</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Sigma</td>
			<td>E7023-1L</td>
			<td>RNA extraction</td>
		</tr>
		<tr>
			<td>iScript cDNA Synthesis Kit</td>
			<td>BioRad</td>
			<td>170-8891</td>
			<td>Reverse transcription</td>
		</tr>
		<tr>
			<td>iTaq Universal SYBR Green Supermix</td>
			<td>BioRad</td>
			<td>175-5122</td>
			<td>Quantative PCR</td>
		</tr>
		<tr>
			<td>miRNeasy Micro Kit (50)</td>
			<td>Qiagen</td>
			<td>217084</td>
			<td>Kit for total RNA isolation</td>
		</tr>
		<tr>
			<td>miScript II RT Kit (50)</td>
			<td>Qiagen</td>
			<td>218161</td>
			<td>Kit for miRNA reverse transcription</td>
		</tr>
		<tr>
			<td>miScript Sybr Green PCR Kit (200)</td>
			<td>Qiagen</td>
			<td>218073</td>
			<td>Kit for real-time PCR expression analysis of miRNAs</td>
		</tr>
		<tr>
			<td>Centrifuge 5424R</td>
			<td>Eppendorf</td>
			<td></td>
			<td>For centrifugation of 1.5ml or 2.0ml tubes at different temperatures. Max speed - 21130g</td>
		</tr>
		<tr>
			<td>Centrifuge 5810R</td>
			<td>Eppendorf</td>
			<td></td>
			<td>For real-time PCR plate centrifugation at different temperatures. Max speed - 2039g</td>
		</tr>
		<tr>
			<td>My Cycler Thermal Cycler</td>
			<td>Bio-Rad</td>
			<td></td>
			<td>For reverse transcription</td>
		</tr>
		<tr>
			<td>CFX96 Real-Time System/C1000 Touch Thermal Cycler</td>
			<td>Bio-Rad</td>
			<td></td>
			<td>For real-time PCR analysis</td>
		</tr>
		<tr>
			<td>miRNeasy Serum/Plasma Spike-in Control</td>
			<td>Qiagen</td>
			<td>219610</td>
			<td>For quality control of RNA isolation</td>
		</tr>
		<tr>
			<td>Hard-Shell 96-Well PCR Plates, low profile, thin wall, skirted, green/clear</td>
			<td>Bio-Rad</td>
			<td>HSP9641</td>
			<td>For real-time PCR analysis</td>
		</tr>
		<tr>
			<td>Microseal &#39;B&#39; PCR Plate Sealing Film, adhesive, optical</td>
			<td>Bio-Rad</td>
			<td>MSB1001</td>
			<td>For real-time PCR plate sealing</td>
		</tr>
		<tr>
			<td>Research plus Single-Channel Pipette, Gray; 0.5-10 &#181;L</td>
			<td>Eppendorf</td>
			<td>UX-24505-02</td>
			<td>For pipetting</td>
		</tr>
		<tr>
			<td>PIPETMAN Classic Pipets, P20</td>
			<td>Gilson</td>
			<td>F123600G</td>
			<td>For pipetting</td>
		</tr>
		<tr>
			<td>PIPETMAN Classic Pipets, P200</td>
			<td>Gilson</td>
			<td>F144565</td>
			<td>For pipetting</td>
		</tr>
		<tr>
			<td>Rainin L-1000XLS Pipet-Lite XLS LTS Pipette 100-1000 &#181;L</td>
			<td>Gilson</td>
			<td>17011782</td>
			<td>For pipetting</td>
		</tr>
		<tr>
			<td>Glycogen, RNA grade</td>
			<td>Thermo Fisher Scientific</td>
			<td>R0551</td>
			<td>Improves total RNA isolation efficiency</td>
		</tr>
		<tr>
			<td>Posi-Click 1.7 mL Tubes, natural color</td>
			<td>Denville</td>
			<td>C2170</td>
			<td>RNA isolation and storage; reagent mix</td>
		</tr>
		<tr>
			<td>Thermal Cycling Tubes -0.2 mL Individual Caps, Standard 0.2 mL tubes with optically</td>
			<td>Denville</td>
			<td>C18098-4 (1000910)</td>
			<td>Reverse transcription reaction</td>
		</tr>
		<tr>
			<td>Sharp 10 Precision barrier Tips</td>
			<td>Denville</td>
			<td>P1096-FR</td>
			<td>For pipetting</td>
		</tr>
		<tr>
			<td>Sharp 20 Precision barrier Tips</td>
			<td>Denville</td>
			<td>P1121</td>
			<td>For pipetting</td>
		</tr>
		<tr>
			<td>Sharp 200 Precision barrier Tips</td>
			<td>Denville</td>
			<td>P1122</td>
			<td>For pipetting</td>
		</tr>
		<tr>
			<td>Tips LTS 1 mL Filter</td>
			<td>Rainin</td>
			<td>RT-L1000F</td>
			<td>For pipetting</td>
		</tr>
		<tr>
			<td>miScript Primer Assay (200)</td>
			<td>Qiagen</td>
			<td>(it changes according to the miRNA)</td>
			<td>For real-time PCR analysis</td>
		</tr>
		<tr>
			<td>Gradient Master ver 5.3 Model 108</td>
			<td>BioComp Instruments</td>
			<td></td>
			<td>For preparation of sucrose gradients</td>
		</tr>
		<tr>
			<td>trichloroacetic acid</td>
			<td>Sigma Aldrich</td>
			<td>T6399</td>
			<td></td>
		</tr>
		<tr>
			<td>acetone</td>
			<td>Sigma Aldrich</td>
			<td>650501</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris hydrochloride</td>
			<td>Amresco</td>
			<td>M108</td>
			<td></td>
		</tr>
		<tr>
			<td>dithiothreitol</td>
			<td>Fisher Scientific</td>
			<td>BP172</td>
			<td></td>
		</tr>
		<tr>
			<td>iodoacetamide</td>
			<td>Gbiosciences</td>
			<td>RC-150</td>
			<td></td>
		</tr>
		<tr>
			<td>sequencing grade modified trypsine, porcine</td>
			<td>Promega</td>
			<td>V5111</td>
			<td></td>
		</tr>
		<tr>
			<td>ammonium bicarbonate</td>
			<td>BDH</td>
			<td>BDH9206</td>
			<td></td>
		</tr>
		<tr>
			<td>formic acid, Optima LC/MS</td>
			<td>Fisher Chemical</td>
			<td>A117</td>
			<td></td>
		</tr>
		<tr>
			<td>methanol, Optima LC/MS</td>
			<td>Fisher Chemical</td>
			<td>A454</td>
			<td></td>
		</tr>
		<tr>
			<td>acetonitrile, Optima LC/MS</td>
			<td>Fisher Chemical</td>
			<td>A996</td>
			<td></td>
		</tr>
		<tr>
			<td>Protein LoBind tubes 0.5 mL</td>
			<td>Eppendorf AG</td>
			<td>22431064</td>
			<td></td>
		</tr>
		<tr>
			<td>Protein LoBind tubes 1.5 mL</td>
			<td>Eppendorf AG</td>
			<td>22431081</td>
			<td></td>
		</tr>
		<tr>
			<td>HLB &#181;Elution plate 30 &#181;m</td>
			<td>Oasis</td>
			<td>186001828BA</td>
			<td></td>
		</tr>
		<tr>
			<td>SpeedVac concentrator</td>
			<td>Thermo Scientific</td>
			<td>Savant SPD2010</td>
			<td></td>
		</tr>
		<tr>
			<td>sonicator</td>
			<td>Qsonica</td>
			<td>Oasis180</td>
			<td></td>
		</tr>
		<tr>
			<td>centrifuge</td>
			<td>Thermo Scientific</td>
			<td colspan="2">Sorvall Legend micro 21R</td>
		</tr>
		<tr>
			<td>LC trap column PepMap 100 C18</td>
			<td>Thermo Scientific</td>
			<td>160454</td>
			<td></td>
		</tr>
		<tr>
			<td>LC separation column PepMap RSLC C18</td>
			<td>Thermo Scientific</td>
			<td>164536</td>
			<td></td>
		</tr>
		<tr>
			<td>mass spectrometer</td>
			<td>Thermo Scientific</td>
			<td colspan="2">Orbitrap Elite ion trap mass spectrometer</td>
		</tr>
		<tr>
			<td>MSConvert software</td>
			<td>ProteoWizard Toolkit</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sorcerer-SEQUEST software</td>
			<td>Sage-N Research, Inc.</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

dynamic proteomic and mirna analysis of polysomes from isolated mouse heart after langendorff perfusion

Estudos em mudanças dinâmicas na tradução de proteínas requerem métodos especializados. Aqui, examinamos as alterações em proteínas recém sintetizadas em resposta à isquemia e reperfusão, usando o coração isolado de rato perfundido juntamente com polysome de criação de perfil. Para se perceber as mudanças dinâmicas na tradução de proteína, caracteriza-se os mRNAs que foram carregados com ribossomas citosólico (poliribosomami ou polissomos) e também recuperou polissomos mitocondriais e comparado a distribuição do mRNA e proteína na frações de alta eficiência (numerosos ribossomos mRNA), baixo-eficiência (menos ribossomos), que também incluiu polissomos mitocondriais e as frações não-traduzindo. os miRNAs pode também associar mRNAs que estão sendo traduzidos, reduzindo assim a eficiência da tradução, examinamos a distribuição dos miRNAs em frações. A distribuição de mRNAs miRNAs e proteínas foi examinada sob condições perfundidas basais, no final de 30 min de isquemia global de não-fluxo e após 30 min de reperfusão. Aqui apresentamos os métodos usados para realizar esta análise — em particular, a abordagem de otimização de extração da proteína do gradiente de sacarose, como este não tem sido descrita antes — e fornecer alguns resultados representativos.

análise do mRNA resultados de mRNA podem ser expressa como uma distribuição de um mRNA específico em cada fração (Figura 3A); para quantificação, combinar polyribosomal frações de traduzir e comparar com a fração não-traduzindo (Figura 3B), apresentando uma relação de abundância de mRNA em traduzir para frações nontranslating. Informações adicionais de são adquiridas, examinando as frações de po...

Watch this Scientific Journal Video about Dynamic Proteomic and miRNA Analysis of Polysomes from Isolated Mouse Heart After Langendorff Perfusion at JoVE.com

Dynamic Proteomic and miRNA Analysis of Polysomes from Isolated Mouse Heart After Langendorff Perfusion

Aqui nós apresentamos um protocolo para realizar polysome de criação de perfil em coração isolado de rato perfundido. Nós descrevemos os métodos para análise das frações no que diz respeito a mRNAs, miRNAs e o proteome polysome polysome, polysome de criação de perfil e perfusão do coração.

Dinâmica proteómica e miRNA análise de polissomos de coração de rato isolado após Langendorff perfusão

Studies in dynamic changes in protein translation require specialized methods. Here we examined changes in newly-synthesized proteins in response to ...

Research

JoVE Journal

Biochemistry

Resolving Affinity Purified Protein Complexes by Blue Native PAGE and Protein Correlation Profiling

Resolver purificada por afinidade complexos de proteínas por Azul Native PAGE e Proteína Correlação Profiling

Most proteins act in association with others; hence, it is crucial to characterize these functional units in order to fully understand biological ...

Bioquímica

Dissection of Human Retina and RPE-Choroid for Proteomic Analysis

Dissecação da Retina humana e RPE coroide para análise proteômica

The human retina is composed of the sensory neuroretina and the underlying retinal pigmented epithelium (RPE), which is firmly complexed to the vascular ...

Purification of Hepatocytes and Sinusoidal Endothelial Cells from Mouse Liver Perfusion

Purificação de hepatócitos e células endoteliais sinusoidais de fígado de rato perfusão

This protocol demonstrates a method for obtaining high yield and viability for mouse hepatocytes and sinusoidal endothelial cells (SECs) suitable for ...

Split-BioID &#8212; Proteomic Analysis of Context-specific Protein Complexes in Their Native Cellular Environment

Split-BioID ÃƒÆ’Ã‚Â¢ÃƒÂ¢Ã¢â‚¬Å¡Ã‚Â¬ÃƒÂ¢Ã¢â€šÂ¬Ã‚Â Proteomic Analysis of Context-specific Protein Complexes in Their Native Cellular Environment

Split-BioID — Proteomic análise de complexos de proteínas específicas do contexto em seu ambiente nativo do celular

To complement existing affinity purification (AP) approaches for the identification of protein-protein interactions (PPI), enzymes have been introduced ...

Analyzing Dynamic Protein Complexes Assembled On and Released From Biolayer Interferometry Biosensor Using Mass Spectrometry and Electron Microscopy

Analisando a dinâmica da proteína montada sobre e libertado da interferometria Biolayer Biosensor usando espectrometria de massa e de microscopia eletrônica

In vivo, proteins are often part of large macromolecular complexes where binding specificity and dynamics ultimately dictate functional outputs. In this ...

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

Livre de calibração em Vitro quantificação da proteína Homo-oligomerização usando instrumentação comercial e livre, Open Source Software de análise de brilho

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

Proteomic Analysis of Human Macrophage Polarization Under a Low Oxygen Environment

Análise de Proteomic do macrófago humano polarização sob um ambiente de baixo oxigênio

Macrophages are innate immune cells involved in a number of physiological functions ranging from responses to infectious pathogens to tissue homeostasis. ...

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

Perfil de Ubiquitin e Ubiquitin-like Modificações pós-translacionais dependentes e identificação de alterações significativas

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

Proteomic Profile of EPS-Urine through FASP Digestion and Data-Independent Analysis

Perfil proteômico de EPS-urina através da Digestão FASP e Análise Independente de Dados

Filter-aided sample protocol (FASP) is widely used for proteomics sample preparation because it allows to concentrate diluted samples and it is compatible ...

F&#246;rster Resonance Energy Transfer Measurements in Living Plant Cells

FÃƒÆ’Ã‚Â¶rster Resonance Energy Transfer Measurements in Living Plant Cells

Medidas de transferência de energia de ressonância förster em células vegetais vivas

Sensitized emission-based F&#246;rster resonance energy transfer (FRET) experiments are easily done but depend on the microscopic setup. Confocal laser ...