En modificeret Nedbør fremgangsmåde til isolering Urinary Exosomer
Summary
This manuscript describes a protocol for isolating exosomes from human urine using a modified precipitation technique.
Abstract
Identification of biomarkers that allow early detection of kidney diseases in urine and plasma has been an area of active interest for several years. Urinary exosome vesicles, 40-100 nm in size, are released into the urine under normal conditions by cells from all nephron segments and may contain protein, mRNA and microRNA representative of their cell type of origin. Under conditions of renal dysfunction or injury, exosomes may contain altered proportions of these components, which may serve as biomarkers for disease. There are currently several methods available for isolation of urinary exosomes, and we have previously conducted an experimental comparison of each of these approaches, including three based on ultracentrifugation, one using a nanomembrane ultrafiltration concentrator, one using a commercial precipitation reagent and one using a modification of the precipitation technique using ExoQuick reagent that we developed in our laboratory. We found the modified precipitation method produced the highest yield of exosome particles, miRNA, and mRNA, making this approach suitable for the isolation of exosomes for subsequent RNA profiling. We conclude that the modified exosome precipitation method offers a quick, scalable, and effective alternative for the isolation of exosomes from urine. In this report, we describe our modified precipitation technique using ExoQuick reagent for isolating exosomes from human urine.
Introduction
Urinary exosomer er små interne vesikler på 100 nm af multivesikulære organer (MVB) afledt af alle celletyper under normale og patologiske forhold i urin rum herunder glomerulære podocytter, nyretubuli og celler foring urin drænsystemer, der synes at blive beriget for miRNA 1 -2. Mange forskere har opdaget forskellige proteiner i exosomer isoleret fra urin fra raske individer såvel som dem med nyre- og / eller systematiske sygdomme 3-5. Exosomer kan isoleres ved hjælp af forskellige metoder, såsom totrins forskellen ultracentrifugering med eller uden saccharose 6-7 kombinere nanomembrane filter med ultracentrifugering 8-9, eller udfældning 10-11. Vi har for nylig udviklet en enkel, hurtig, skalerbar og effektiv metode til at isolere urin exosomer og opdage miRNA og protein biomarkører 11. Selv biomarkører kan påvises i en række forskellige biologiske fluider, urIne er den mest bekvemme valg for patienter med sygdomme i nyrerne og urinvejene, fordi det kan opnås i store mængder i en relativt ikke-invasiv måde.
Selv om der er flere metoder til at isolere urin exosomer 6-11, den mest anvendte procedure er baseret på forskellen ultracentrifugering med en saccharosepude 30%. Selv om denne fremgangsmåde er effektiv og kvaliteten af de resulterende exosomer isoleret med denne procedure er høj, selve teknikken kræver uddannet personale og er tidskrævende, kræver adskillige ultracentrifugering trin. Andre metoder, såsom filtrering og udfældning, er blevet udviklet til isolering af exosomer, herunder en, der bruger en navnebeskyttet udfældningsreagens (dvs. ExoQuick-TC: System Biosciences; Mountain View, CA). Selv udfældning ved hjælp af dette reagens er hurtig og relativt let, renheden af exosomer isoleret er lavt i forhold til ultracentrifugering method. Vi har for nylig sammenlignet seks metoder til isolering af urin exosomer, herunder en modifikation af nedbør under anvendelse af ExoQuick-TC, som vi har udviklet i vores laboratorium 11. Vi fandt, at dette ændrede nedbør metode gav større mængder af protein, miRNA og mRNA og selve proceduren var hurtigere og lettere at implementere end ultracentrifugering. Her beskriver vi forsøgsprotokollen af vores ændrede nedbør metode på en trinvis måde, og inkluderer en diskussion af fordele og ulemper ved denne teknik sammenlignet med andre metoder til exosome isolation. Den modificerede nedbør fremgangsmåde involverer en indledende udfældning af exosom-partikler, efterfulgt af fjernelse af Tamm-Horsfall protein, som er korreleret med exosomal proteiner og kendt for at reducere exosome udbytte fra urin. Vi fandt, at disse ændringer øget udbytte og renhed af præparatet exosomal fra urin.
Protocol
Representative Results
Discussion
De fleste metoder med udfældning af exosomer fra urin ikke fat fjernelse af det polymere netværk dannet af Tamm-Horsfall protein (BBE). Dette protein er til stede i store mængder i urinen, når den -sagerne fælder exosomer under indledende lavhastighedscentrifugering. I vores modificeret fremgangsmåde vi reduceret THP hjælp DTT før exosome udfældning. Som vist i figur 2, blev en større mængde af THP observeret i uforandret urin og pellet, med ingen THP i den endelige supernatant og exosome pel…
Disclosures
The authors have nothing to disclose.
Acknowledgements
We are grateful to the Roney Family Foundation for the support of this study. We acknowledge the contribution of Dr. M. Lucrecia Alvarez to the planning and execution of the experiments described in this work.
Materials
| Name of Material/ Equipment | Company | Catalog Number | Comments/Description |
| Corning 15ml Centrifuge Tube | Corning (Sigma Aldrich) | CLS430791 | |
| Protease Inhibitor Cocktail | Sigma Aldrich | P8340 | |
| Centrifuge | Sorvall | ||
| DL-dithiothreitol | Sigma Aldrich | D9779 | used at final concentration of 200 mg/ml |
| Novex NuPAGE SDS-PAGE system (4-12 % Bis-Tris Gel 1.0 mm, 10 well | Invitrogen | NP0321BOX | For SDS-PAGE analysis |
| ECL Advance Western blotting detection kit | GE Healthcare Life Sciences | RPN2135 | For western blot detection of biomarkers |
| Exoquick-TC Reagent | System Biosciences (SBI) | EXOTC50A-1 | For exosome isolation |
| Exosome CD9 ELISA Complete Kit | System Biosciences (SBI) | EXOEL-CD9-A-1 | For exosome quantification |
| AllPrep DNA/RNA/Protein mini kit | Qiagen | 80004 | For DNA, RNA, Protein isolation |
| Rneasy MinElute Cleanup kit | Qiagen | 74204 | |
| NanoDrop 2000 UV-Vis Spectrophotometer | Thermo Scientific | For Protein Quantification | |
| ProteoSilver Sliver Stain Kit | Sigma Aldrich | PROTSIL1-1KT | For staining SDS-PAGE gels |
| Xcell SureLock Mini-Cell Electrophoresis System | Life Technologies | For running the SDS-PAGE gels | |
| TaqMan microRNA assays | Life Technologies | For RT-PCR assays | |
| qBasePlus v1.5 software | Biogazelle NV | For analysis of RT-PCR assay data | |
| Rabbit polyclonal antibody to ALIX | Millipore | For western blot detection of biomarkers | |
| Mouse monoclonal antibody to TSG101 | Abcam | For western blot detection of biomarkers |
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