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 (straccio, JVE), NIH R01 HL132075 (straccio, JVE), centro Barbra Streisand femminile del cuore (straccio, JVE), Dorothy ed E. Phillip Lione sedia in cardiologia molecolare (RAG), Erika Glazer dotato sedia in salute del cuore della donna (JVE) e Accademia Ceca delle scienze Sostegno istituzionale RVO: 68081715 (MS).

Tutti gli studi sugli animali sono stati effettuati in conformità alle linee guida istituzionali e approvati dalla istituzionale Animal Care e utilizzare Comitato del Cedars-Sinai Medical Center.
1. Langendorff aspersione del Mouse cuore
<ol><li>
Aspersione di Langendorff del cuore del topo con ischemia e riperfusione
	<ol>
<li>Amministrare intraperitoneale sodio pentobarbital 70 mg/kg per il topo adulto (8-settimana-vecchio, Maschio, C57BL6/j). Confermare l'anestesia profo...

<ol>
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Analisi del profilo polisoma consente lo studio della traduzione della proteina analizzando lo stato traduzionale di un mRNA specifico o l'intero trascrittoma6,7. Inoltre è di grande aiuto quando traduzione locale deve essere studiato come sinaptosomi8. Tradizionalmente, questo metodo comporta la separazione di mono - e poliribosomi e i mRNAs associati su un gradiente di saccarosio che potrebbe essere accoppiato con genomica o tecniche di...

Il cuore risponde al pregiudizio di ischemia (I) e di riperfusione (R) in modo dinamico. Tuttavia, non esiste una visione poco in cambiamenti acuti nella sintesi delle proteine durante la risposta. Per risolvere questo problema, abbiamo approfittato del metodo consolidato di polisoma profilatura1 per identificare le modifiche in abbondanza di proteine che riflettono la ridistribuzione dei ribosomi e fattori di regolazione traduzionale dal citosol di polisomi, e la aumento di proteine recentemente sintetizzati (NSP). Nell'impostazione di I / R, l'aumento nella sintesi di nuove proteine avviene in un lasso di tempo che non è coerente con la trascrizione di mRNA nuovo2; Inoltre, discordanza tra i livelli di espressione di mRNA e proteina abbondanza è stato segnalato3. Per queste ragioni, abbiamo scelto di analizzare i cambiamenti nel proteoma dinamico come riflesso dalla traduzione della proteina. Per fare questo, abbiamo quantificare mRNA nelle frazioni polisoma e analizzare la composizione della proteina nelle frazioni polisoma. Infine, perché i microRNA (miRs) regolano la disponibilità dei mRNAs per traduzione e possono interferire con l'efficienza di proteina traduzione4,5, abbiamo esaminato la distribuzione di Mir nelle frazioni polisoma, concentrandosi sulla risposta a I / r.
Abbiamo scelto di utilizzare il modello di aspersione di isolato mouse Langendorff e tessuto raccolto in condizioni basali della perfusione continua, dopo ischemia globale di no--flusso 30 min e dopo 30 min di ischemia seguita da 30 min di riperfusione. Abbiamo poi solubilizzato il tessuto cardiaco e separato polisomi sopra un gradiente di saccarosio, seguita da analisi proteomica e rivelazione selettiva del mRNA e Mirna mediante PCR e microarray, rispettivamente. Questa combinazione di metodi rappresenta un approccio potente per comprendere il proteoma dinamico, consentendo la rilevazione simultanea di mRNA, miRNA e NSP, così come la ridistribuzione delle proteine regolatrici, miRNA e mRNA tra le frazioni di nontranslating, bassa efficienza polisomi e ad alta efficienza polisomi (Vedi Figura 1). Intuizioni la regolazione dinamica di questo processo saranno esteso da un'ulteriore analisi della fosforilazione di fattori regolatori chiave quali eIF2α o mTOR. Questi singoli passi ora sono descritti dettagliatamente.

<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

Gli studi in cambiamenti dinamici nella traduzione della proteina richiedono metodi specializzati. Qui abbiamo esaminato i cambiamenti in proteine appena sintetizzate in risposta all'ischemia e riperfusione usando il cuore isolato e perfuso del mouse accoppiato con profilatura polisoma. Per capire meglio i cambiamenti dinamici nella traduzione della proteina, abbiamo caratterizzato i mRNAs che sono stati caricati con i ribosomi citosolici (poliribosomi o polisomi) e anche recuperato polisomi mitocondriale e rispetto distribuzione mRNA e proteina nella le frazioni ad alta efficienza (numerosi ribosomi attaccati al mRNA), basso-efficienza (meno ribosomi allegati) che comprendeva anche polisomi mitocondriale e le frazioni non tradurre. Mirna possono anche associare mRNAs che vengono tradotti, riducendo così l'efficienza della traduzione, abbiamo esaminato la distribuzione dei miRNA attraverso le frazioni. La distribuzione dei mRNAs, Mirna e proteine è stata esaminata in condizioni basali irrorate, alla fine di 30 min di ischemia globale no-flusso e dopo 30 min di riperfusione. Qui presentiamo i metodi utilizzati per realizzare questa analisi — in particolare, l'approccio all'ottimizzazione dell'estrazione di proteine dal gradiente di saccarosio, come questo non è stato descritto prima — e fornire alcuni risultati rappresentativi.

analisi del mRNA risultati di mRNA possono essere espressa come una distribuzione di un particolare mRNA in ogni frazione (Figura 3A); per la quantificazione, combinare polyribosomal traduzione frazioni e confrontarlo con la frazione non-tradurre (Figura 3B), che presentano un rapporto di abbondanza del mRNA nella traduzione al nontranslating frazioni. Ulteriori informazioni sono maturate esaminando le frazioni di alt...

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

Qui presentiamo un protocollo per eseguire polisoma profilatura sul cuore isolato e perfuso del mouse. Descriviamo i metodi per aspersione di cuore, polisoma profiling e l'analisi delle frazioni polisoma rispetto al mRNA, Mirna e il proteoma polisoma.

Dinamica proteomica e miRNA analisi di polisomi da Mouse cuore dopo Langendorff aspersione isolata

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

Risoluzione purificato per affinità complessi proteici di Blue Native PAGE e proteine ​​correlazione Profiling

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

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Biochimica

Dissection of Human Retina and RPE-Choroid for Proteomic Analysis

Dissezione della Retina umana e RPE-coroide per analisi proteomica

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

Purificazione di epatociti e cellule endoteliali sinusoidali da fegato di topo perfusione

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 — Analisi proteomica dei complessi della proteina contesto specifico in ambiente nativo di cellulare

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

L'analisi di complessi proteici dinamico montato su e rilasciato da Biolayer interferometria biosensore utilizzando la microscopia elettronica e spettrometria di massa

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

Privo di calibrazione In Vitro quantificazione delle proteine omo-oligomerizzazione utilizzando Strumentazione commerciale e libero, Open Source Software di analisi di luminosità

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

Analisi proteomica di polarizzazione di macrofago umano in un ambiente a basso tenore di ossigeno

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

Profilazione delle modifiche post-traduzionali dipendenti da Ubiquitin e Ubiquitine e identificazione di alterazioni significative

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

Profilo proteomico dell'EPS-Urina attraverso la digestione FASP e l'analisi indipendente dai dati

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

Misurazioni del trasferimento di energia di risonanza di Förster in cellule vegetali viventi

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