Dynamisk proteom og miRNA analyse af Polysomes fra isolerede mus hjertet efter Langendorff Perfusion
Summary
Her præsenterer vi en protokol for at udføre polysome profilering på isolerede perfunderet mus hjerte. Vi beskriver metoder til hjertet perfusion, polysome profilering og analyse af polysome fraktioner mRNAs, miRNAs og polysome proteomet.
Abstract
Undersøgelser i dynamiske ændringer i protein oversættelse kræver specialiserede metoder. Her undersøgte vi ændringer i nyligt syntetiseret proteiner i svar til iskæmi og reperfusion ved hjælp af isolerede perfunderet mus hjertet kombineret med polysome profilering. For yderligere at forstå de dynamiske ændringer i protein oversættelse, vi kendetegnet den mRNAs, der var lastet med cytosole ribosomer (polyribosomes eller polysomes) og også tilbagebetalte mitokondrie polysomes og sammenlignet mRNA og protein fordeling i de højeffektiv fraktioner (mange ribosomer knyttet til mRNA), lav-effektivitet (færre ribosomer vedlagt), der også omfattede mitokondrie polysomes, og de ikke-oversætte fraktioner. miRNAs kan også knytte mRNAs, der er ved at blive oversat, hvorved effektiviteten af oversættelse, vi undersøgte distribution af miRNAs på tværs af fraktioner. Fordelingen af mRNAs, miRNAs og proteiner blev undersøgt på basal perfunderet betingelser, i slutningen af 30 min globale no-flow iskæmi, og efter 30 min af reperfusion. Her præsenterer vi de metoder, der anvendes til at udføre denne analyse — navnlig tilgang til optimering af protein udvinding fra saccharose gradient, da dette er ikke blevet beskrevet før — og give nogle repræsentative resultater.
Introduction
Hjertet reagerer på skade af iskæmi (I) og reperfusion (R) på en dynamisk måde. Der er dog lidt indsigt i akutte ændringer i proteinsyntesen i svaret. For at løse dette, tog vi fordel af den veletablerede metode af polysome profilering1 for at identificere ændringer i protein overflod, der afspejler omfordeling af ribosomer og translationel regulerende faktorer fra cytosol til polysomes, og den forøgelse af nyligt syntetiserede proteiner (NSP). I fastsættelsen af jeg / R, stigning i nye proteinsyntesen opstår i en tidsramme, der er i strid med transskription af nye mRNAs2; Derudover har uoverensstemmelse mellem mRNA udtryk niveauer og protein overflod været rapporteret3. Af disse årsager valgte vi at analysere ændringer i det dynamiske proteomet som reflekteres af protein oversættelse. For at gøre dette, vi kvantificere mRNA i polysome fraktioner, og analysere protein sammensætning i polysome fraktioner. Endelig fordi MicroRNA (miRs) regulere tilgængeligheden af mRNAs for oversættelse og kan forstyrre effektivitet af protein oversættelse4,5, vi undersøgte distribution af miRs i polysome brøker, med fokus på den svar på jeg / R.
Vi valgte at bruge isoleret mus Langendorff perfusion model og høstede væv under basale forhold af kontinuerlig perfusion, efter 30 min globale no-flow iskæmi, og efter 30 min for iskæmi efterfulgt af 30 min. af reperfusion. Vi derefter solubilized hjertet væv og adskilt polysomes over en saccharose gradient, efterfulgt af proteom analyse og selektiv påvisning af mRNAs og miRNAs af PCR og microarray, henholdsvis. Denne kombination af metoder repræsenterer en kraftfuld tilgang til forståelsen af dynamiske proteomet, så samtidige påvisning af mRNA, miRNA, og NSP, omfordeling af regulerede proteiner, miRNA og mRNA mellem nontranslating fraktioner, ringe virkningsgrad polysomes, og højeffektiv polysomes (Se figur 1). Indsigt i den dynamiske regulering af denne proces vil blive udvidet med yderligere analyse af fosforylering af centrale lovgivningsmæssige faktorer såsom eIF2α eller mTOR. Disse individuelle skridt er nu beskrevet i detaljer.
Protocol
Representative Results
Discussion
Polysome profil analyse giver mulighed for undersøgelse af protein oversættelse ved at analysere et bestemt mRNA eller hele transkriptom6,7translationel tilstand. Det er også af stor hjælp når lokale oversættelse skal studeres som synaptosomes8. Traditionelt, indebærer denne metode adskillelse af mono – og polyribosomes og den tilknyttede mRNAs på et saccharose forløb, som kan kobles til genomisk eller proteom teknikker til at opn…
Declarações
The authors have nothing to disclose.
Acknowledgements
NIH P01 HL112730 (RAG, JVE), NIH R01 HL132075 (RAG, JVE), Barbra Streisand kvinders Heart Center (RAG, JVE), Dorothy og E. Phillip Lyon stol i molekylær kardiologi (RAG), Erika Glazer begavet stol i kvindens hjertesundhed (JVE) og tjekkiske videnskabsakademi Institutionel støtte RVO: 68081715 (MS).
Materials
| Pentobarbital | Vortech Phamaceuticals | 9373 | for euthansia |
| Heparin | Sagent | 103424 | used in langendorff preparation |
| forceps | Fine Science Tools | 91110-10 | used to hang the heart |
| Langendorff system | Radnoti + home made | n/a | A 'four heart' system consisting of custom blown glass, tubing and water baths |
| NaCl | Sigma | S7653-5KG | Krebs buffer and Sucrose gradient |
| KCl | Sigma | P5405 | Krebs buffer and Lysis buffer |
| KH2PO4 | Sigma | P-5504 | Krebs buffer |
| MgSO4 | Sigma | M7774-500G | Krebs buffer |
| Glucose | Sigma | G5767 | Krebs buffer |
| CaCl2 | Sigma | C1016-500G | Krebs buffer |
| Sucrose powder | Sigma | S0389-1KG | Sucrose gradient |
| MgCl2 | Sigma | 208337 | Sucrose gradient and Lysis buffer |
| Tris-base | Sigma | T1503-1KG | Sucrose gradient and Lysis buffer |
| Xylene Cyanole | Sigma | X-4126 | Sucrose gradient |
| Cycloheximide | Sigma-aldrich | 239763 | Sucrose gradient and Lysis buffer |
| RNaseOUT | Life Technologies | C00019 | RNAse inhibitor for Lysis buffer |
| Igepal CA-360 (NP40) | Sigma | I3021 | Lysis buffer |
| Protease Inhibitor Cocktail tablets, EDTA free | Roche | 5056489001 | |
| Tube, Thinwall, Ultra-Clear, 13.2 mL, 14 x 89 mm | Beckman Coulter | 344059 | |
| Ultracentrifuge | Beckman | LE-80K | Ultracentrifugation of the gradients |
| Rotor | Beckman | SW41 | Ultracentrifugation of the gradients |
| Biologic LP (pump) | Biorad | 731-8300 | Fractionation of the gradients |
| BioFrac | Biorad | 741-0002 | Fractionation of the gradients |
| Eppendorf RNA/DNA LoBind microcentrifuge tubes, 2 mL tube | Sigma | Z666513-100EA | Gradient fraction and RNA extraction |
| TRIzol Reagent | Life technologies | AM9738 | RNA extraction |
| Luciferase Control RNA | Promega | L4561 | RNA extraction |
| Chloroform | Fisher Scientific | C606-4 | RNA extraction |
| Glycogen, RNA grade | Thermo Fisher Scientific | R0551 | RNA extraction |
| Isopropanol | Sigma | I9516 | RNA extraction |
| Ethanol | Sigma | E7023-1L | RNA extraction |
| iScript cDNA Synthesis Kit | BioRad | 170-8891 | Reverse transcription |
| iTaq Universal SYBR Green Supermix | BioRad | 175-5122 | Quantative PCR |
| miRNeasy Micro Kit (50) | Qiagen | 217084 | Kit for total RNA isolation |
| miScript II RT Kit (50) | Qiagen | 218161 | Kit for miRNA reverse transcription |
| miScript Sybr Green PCR Kit (200) | Qiagen | 218073 | Kit for real-time PCR expression analysis of miRNAs |
| Centrifuge 5424R | Eppendorf | For centrifugation of 1.5ml or 2.0ml tubes at different temperatures. Max speed – 21130g | |
| Centrifuge 5810R | Eppendorf | For real-time PCR plate centrifugation at different temperatures. Max speed – 2039g | |
| My Cycler Thermal Cycler | Bio-Rad | For reverse transcription | |
| CFX96 Real-Time System/C1000 Touch Thermal Cycler | Bio-Rad | For real-time PCR analysis | |
| miRNeasy Serum/Plasma Spike-in Control | Qiagen | 219610 | For quality control of RNA isolation |
| Hard-Shell 96-Well PCR Plates, low profile, thin wall, skirted, green/clear | Bio-Rad | HSP9641 | For real-time PCR analysis |
| Microseal 'B' PCR Plate Sealing Film, adhesive, optical | Bio-Rad | MSB1001 | For real-time PCR plate sealing |
| Research plus Single-Channel Pipette, Gray; 0.5-10 µL | Eppendorf | UX-24505-02 | For pipetting |
| PIPETMAN Classic Pipets, P20 | Gilson | F123600G | For pipetting |
| PIPETMAN Classic Pipets, P200 | Gilson | F144565 | For pipetting |
| Rainin L-1000XLS Pipet-Lite XLS LTS Pipette 100-1000 µL | Gilson | 17011782 | For pipetting |
| Glycogen, RNA grade | Thermo Fisher Scientific | R0551 | Improves total RNA isolation efficiency |
| Posi-Click 1.7 mL Tubes, natural color | Denville | C2170 | RNA isolation and storage; reagent mix |
| Thermal Cycling Tubes -0.2 mL Individual Caps, Standard 0.2 mL tubes with optically | Denville | C18098-4 (1000910) | Reverse transcription reaction |
| Sharp 10 Precision barrier Tips | Denville | P1096-FR | For pipetting |
| Sharp 20 Precision barrier Tips | Denville | P1121 | For pipetting |
| Sharp 200 Precision barrier Tips | Denville | P1122 | For pipetting |
| Tips LTS 1 mL Filter | Rainin | RT-L1000F | For pipetting |
| miScript Primer Assay (200) | Qiagen | (it changes according to the miRNA) | For real-time PCR analysis |
| Gradient Master ver 5.3 Model 108 | BioComp Instruments | For preparation of sucrose gradients | |
| trichloroacetic acid | Sigma Aldrich | T6399 | |
| acetone | Sigma Aldrich | 650501 | |
| Tris hydrochloride | Amresco | M108 | |
| dithiothreitol | Fisher Scientific | BP172 | |
| iodoacetamide | Gbiosciences | RC-150 | |
| sequencing grade modified trypsine, porcine | Promega | V5111 | |
| ammonium bicarbonate | BDH | BDH9206 | |
| formic acid, Optima LC/MS | Fisher Chemical | A117 | |
| methanol, Optima LC/MS | Fisher Chemical | A454 | |
| acetonitrile, Optima LC/MS | Fisher Chemical | A996 | |
| Protein LoBind tubes 0.5 mL | Eppendorf AG | 22431064 | |
| Protein LoBind tubes 1.5 mL | Eppendorf AG | 22431081 | |
| HLB µElution plate 30 µm | Oasis | 186001828BA | |
| SpeedVac concentrator | Thermo Scientific | Savant SPD2010 | |
| sonicator | Qsonica | Oasis180 | |
| centrifuge | Thermo Scientific | Sorvall Legend micro 21R | |
| LC trap column PepMap 100 C18 | Thermo Scientific | 160454 | |
| LC separation column PepMap RSLC C18 | Thermo Scientific | 164536 | |
| mass spectrometer | Thermo Scientific | Orbitrap Elite ion trap mass spectrometer | |
| MSConvert software | ProteoWizard Toolkit | ||
| Sorcerer-SEQUEST software | Sage-N Research, Inc. | ||
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