ver. 4-2011-07-11
Contents
1. 1 If frozen thaw culture media or urine sample on ice 2 Combine 10ml sample 2ml ExoQuick TC 3 Mix well by inversion three times 4 Place at 4 C for overnight at least 12 hours 5 Centrifuge at 1500 x g for 30 minutes 6 Remove supernatant keep exosome pellet 7 Centrifuge at 1500 x g for 5 minutes to remove all traces of fluid take great care not to disturb the pellet 8 Add 200 ul Exosome Binding buffer to exosome pellet and vortex 15 seconds 9 Place at room temperature for 5 minutes to allow complete lysis 10 Exosome protein is now ready for immobilization onto micro titer plate Please refer to the ExoELISA manual for the complete protocol Western blot analysis For Western blotting analysis we recommend resuspending the exosome pellet in 1XRIPA buffer with the appropriate protease inhibitor cocktail SBI offers a Western blot antibody detection kit Catalog ExoAB KIT 1 which includes four exosomal marker antibodies CD63 CD9 CD81 HSP70 and a Goat anti Rabbit IgG HRP conjugated secondary antibody specifically tested for use in exosomal protein analysis Page 4 ver 4 2011 07 11 www systembio com ExoQuick Exosome Precipitation Solution Cat EXOQ__A 1 Cat Description Size Anti CD9 Antibody rabbit anti human with goat anti EXOAB CD9A 1 i 25ul rabbit HRP secondary antibody Anti CD63 Antibody rabbit anti human with goat anti rabbit HRP secondary antibody Anti CD81 Antibody rabbit anti hu2. be seen in the Table below Bio fluid Sample volume ExoQuick volume Serum 250 ul 63 ul Ascites fluid 250 ul 63 ul To isolate exosomes from tissue culture media or urine we recommend using the ExoQuick TC reagent cat EXOTC10A 1 or EXOTCS50A 1 which is a distinct formulation from the original ExoQuick reagent detailed in this manual 888 266 5066 Toll Free 650 968 2200 outside US Page 1 System Biosciences SBI ExoQuick Exosome Precipitation l Overview Exosomes are 40 150 nm membrane vesicles secreted by most cell types in vivo and in vitro Exosomes are found in blood urine amniotic fluid malignant ascite fluids and contain distinct subsets of microRNAs depending upon the tumor from which they are secreted SBI s ExoQuick exosome precipitation reagent makes microRNA and protein biomarker discoveries simple reliable and quantitative Enrich for circulating exosomal microRNAs with ExoQuick User Manual and accurately profile them using SBI s SeraMir qPCR arrays No time consuming ultracentrifugation Less expensive than costly antibodies and beads More effective than any other method Use as little as 100 ul of serum Page 2 or bio fluid Serum or Biofluid g Simple one step precipitation E Supernatant p Exosome pellet ver 4 2011 07 11 ExoQuick Solution Exosome precipitation from 250 ul human serum samples was verified by Western blot using an
3. results After centrifugation the exosomes may appear as a beige or white pellet at the bottom of the vessel Exosome pellets obtained from 10 ml of cerebral spinal fluid using ExoQuick 5 Aspirate supernatant Spin down residual ExoQuick solution by centrifugation at 1500 x g for 5 minutes Remove all traces of fluid by aspiration taking great care not to disturb the precipitated exosomes in pellet 6 Resuspend exosome pellet in 1 10 of original volume using sterile or nuclease free water If the pellet is difficult to resuspend add slightly more water to the pellet to further dilute the salt A Using Precipitated Exosomes for RNA Extraction For RNA extraction we recommend following the protocol outlined in the SeraMir Kit user manual as shown here Catalog RA800A 1 RA805A 1 RA806A 1 RA810A 1 and RA820A 1 Thaw serum sample on ice Combine 500ul serum 120 yl ExoQuick Exosome Mix well by inversion three times Isolation Place at 4 C for 30 minutes and Lysis Centrifuge at 13 000 rpm for 2 minutes Remove supernatant keep exosome pellet Add 350 ul LYSIS Buffer to exosome pellet and vortex 15 seconds Place at room temperature for 5 minutes to allow complete lysis optional add 5ul of SeraMir control RNA spike in cat RA805A 1 ONOORWN gt 9 Add 200uI of 100 Ethanol vortex 10 seconds 10 Assemble spin column and collection tube 11 Transfer all 600ul to spin column 12 Centrifuge at 13 000 rpm for 1 mi
4. with SBI s exosome specific antibody e g CD9 at 1 1000 dilution 5 dry milk in TBS T 15 Wash 3X with TBS T 16 Incubate one hour at room temperature with SBI s Goat anti Rabbit HRP antibody at 1 20 000 dilution 5 dry milk in TBS T 17 Wash 3X with TBS T 18 Incubate blot with chemi luminescence substrate and visualize on film or other imaging equipment OCONOARWN gt 1 1X RIPA buffer contains 25mM Tris HCI pH 7 6 150mM NaCl 1 NP 40 1 sodium deoxycholate 0 1 SDS 2X Laemmli buffer contains 4 SDS 20 glycerol 10 2 mercaptoethanol 0 004 bromphenol blue 0 125 M Tris HCI pH 6 8 Example Data and Applications 1 Protein Yield from Exosomes precipitated with ExoQuick versus other Extraction Methods a 888 266 5066 Toll Free 650 968 2200 outside US Page 5 System Biosciences SBI User Manual 2 0 16 E Chromatography E DynaBeads 1 2 C Ultracentrifugation 5 E ExoQuick 08 o 2 2 04 x Lu Ascites 1 Ascites 2 Ascites 3 b Chromat DynaBeads Ultracent ExoQuick As1 As2 As3 Asl As2 As3 Asl As2 As3 Asl As2 As3 Data courtesy of Dr Douglas Taylor Univ Lousiville KY a The quantity of protein was determined by the Bradford microassay method Bio Rad Laboratories using BSA as a standard b Proteins from each exosome isolate were standardized to the original sample volume and equal volumes were applied per lane of a 12 5 SDS PAGE gel Western immunoblotting was perf
5. A Matsubara S Takeshita T Takizawa T Human villous trophoblasts express and secrete placenta specific microRNAs into maternal circulation via exosomes Biol Reprod 2009 Oct 81 4 717 29 Taylor DD Gercel Taylor C MicroRNA signatures of tumor derived exosomes as diagnostic biomarkers of ovarian cancer Gynecol Oncol 2008 Jul 110 1 13 21 Simpson RJ Lim JW Moritz RL Mathivanan S Exosomes proteomic insights and diagnostic potential Expert Rev Proteomics 2009 Jun 6 3 267 83 Review Vil Technical Support For more information about SBI products and to download manuals in PDF format please visit our web site hitp www systembio com For additional information or technical assistance please call or email us at System Biosciences SBI 265 North Whisman Road Mountain View CA 94043 Phone 650 968 2200 888 266 5066 Toll Free Fax 650 968 2277 E mail General Information info systembio com Technical Support tech systembio com Ordering Information orders systembio com Page 10 ver 4 2011 07 11 www systembio com ExoQuick Exosome Precipitation Solution Cat EXOQ_ A 1 ll Licensing and Warranty Statement Limited Use License Use of the ExoQuick Exosome Precipitation Solution i e the Product is subject to the following terms and conditions If the terms and conditions are not acceptable return all components of the Product to System Biosciences SBI within 7 calendar days Purchase
6. J System Biosciences ExoQuick Exosome Precipitation Solution Cat EXOQ5A 1 Cat EXOQ20A 1 User Manual Store kit at 4 C on receipt A limited use label license covers this product By use of this product you accept the terms and conditions outlined in the Licensing and Warranty Statement ver 4 2011 07 11 contained in this user manual ExoQuick Exosome Precipitation Solution Cat EXOQ_ A 1 Contents l Overview _ i isi 2 Il Exosome Isolation Protocol s3 sesesese 3 III Exosome analysis A RNA analysis occ ccccccccscseececeeeeseececseseeceeeees 4 B Protein analysis ooo ceccscsesesesesesesesesesesesesenens 4 IV Example Data and Applications l enn 7 V ADDOINGIX cissie arenan aaa anaa Ea 11 Vi FRREICrEN COS wins scent anana aaa a a 11 Vil Technical SUPPOrt cccceceee eee ee eee eeeee eee eaeeeeeeeeeeees 13 Vill Licensing and Warranty csceseeeeeeeeeeeeeeeeeeeeeees 14 List of Components Item Catalog Reactions ExoQuick exosome EXOQ20A 1 300 reactions precipitation solution 20 ml ExoQuick exosome EXOQ5A 1 75 reactions precipitation solution 5 ml The ExoQuick kits are shipped at room temperature or on blue ice and should be stored at 4 C upon receipt Properly stored kits are stable for 1 year from the date received The reaction size is based on using 250 ul serum for exosome isolation Examples of precipitating exosomes from various biofluids can
7. and use of any part of the Product constitutes acceptance of the above terms The purchaser of the Product is granted a limited license to use the Product under the following terms and conditions The Product shall be used by the purchaser for internal research purposes only The Product is expressly not designed intended or warranted for use in humans or for therapeutic or diagnostic use The Product may not be resold modified for resale or used to manufacture commercial products without prior written consent of SBI This Product should be used in accordance with the NIH guidelines developed for recombinant DNA and genetic research SBI has pending patent applications related to the Product For information concerning licenses for commercial use contact SBI Purchase of the product does not grant any rights or license for use other than those explicitly listed in this Licensing and Warranty Statement Use of the Product for any use other than described expressly herein may be covered by patents or subject to rights other than those mentioned SBI disclaims any and all responsibility for injury or damage which may be caused by the failure of the buyer or any other person to use the Product in accordance with the terms and conditions outlined herein Limited Warranty SBI warrants that the Product meets the specifications described in this manual If it is proven to the satisfaction of SBI that the Product fails to meet these specifications SBI w
8. ill replace the Product or provide the purchaser with a refund This limited warranty shall not extend to anyone other than the original purchaser of the Product Notice of nonconforming products must be made to SBI within 30 days of receipt of the Product SBI s liability is expressly limited to replacement of Product or a refund limited to the actual purchase price SBI s liability does not extend to any damages arising from use or improper use of the Product or losses associated with the use of additional materials or reagents This limited warranty is the sole and exclusive warranty SBI does not provide any other warranties of any kind expressed or implied including the merchantability or fitness of the Product for a particular purpose SBI is committed to providing our customers with high quality products If you should have any questions or concerns about any SBI products please contact us at 888 266 5066 2011 System Biosciences SBI All Rights Reserved 888 266 5066 Toll Free 650 968 2200 outside US Page 11
9. man with goat anti rabbit HRP secondary antibody Anti Hsp70 Antibody rabbit anti human with goat EXOAB C63A 1 25 ul EXOAB CD81A 1 25 ul ESOL Selene anti rabbit HRP secondary antibody zal ExoAb Antibody Kit CD9 CD63 CD81 Hsp70 EXOAB KIT 1 antibodies rabbit anti human with goat anti rabbit 25 ul each HRP secondary antibody EXOEL CD9A 1 Exosome ELISA Complete Kit CD9 detection 96 reactions EXOEL CD63A 1 Exosome ELISA Complete Kit CD63 detection 96 reactions EXOEL CD81A 1 Exosome ELISA Complete Kit CD81 detection 96 reactions If frozen thaw culture media or urine sample on ice Combine 10 ml sample 2 ml ExoQuick TC Exosome Mix well by inversion three times Isolation and Place at 4 C for overnight at least 12 hours lysis Centrifuge at 1500 x g for 30 minutes Remove supernatant keep exosome pellet Centrifuge at 1500 x g for 5 minutes to remove all traces of fluid take great care not to disturb the pellet Add 200 ul RIPA buffer to exosome pellet and vortex 15 seconds Place at room temperature for 5 minutes to allow complete lysis 10 Add Laemmli buffer with Beta mercaptoethanol and heat at 95 C for 5 minutes 11 Chilled on ice for 5 minutes before loading onto gel 12 Perform standard SDS PAGE electrophoresis and Western transfer onto PVDF membrane 13 Block with 5 dry milk in Tris Buffered Saline 0 05 Tween TBS T for 1 hour 14 Incubate blot overnight at 4 C
10. n 888 266 5066 Toll Free 650 968 2200 outside US Page 9 System Biosciences SBI User Manual PS Martin DB Tewari M Circulating microRNAs as stable blood based markers for cancer detection Proc Natl Acad Sci U S A 2008 Jul 29 105 30 10513 8 Laterza OF Lim L Garrett Engele PW Vlasakova K Muniappa N Tanaka WK Johnson JM Sina JF Fare TL Sistare FD Glaab WE Plasma MicroRNAs as sensitive and specific biomarkers of tissue injury Clin Chem 2009 Nov 55 11 1977 83 Valadi H Ekstr m K Bossios A Sj strand M Lee JJ Lotvall JO Exosome mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells Nat Cell Biol 2007 Jun 9 6 654 9 Pegtel DM Cosmopoulos K Thorley Lawson DA van Eijndhoven MA Hopmans ES Lindenberg JL de Gruijl TD Wordinger T Middeldorp JM Functional delivery of viral miRNAs via exosomes Proc Natl Acad Sci USA 2010 Apr 6 107 14 6328 33 Mathivanan S and Simpson R J ExoCarta A compendium of exosomal proteins and RNA Proteomics 2009 21 4997 5000 Thery C Ostrowski M Segura E Membrane vesicles as conveyors of immune responses Nat Rev Immunol 2009 8 581 93 Michael A Bajracharya SD Yuen PS Zhou H Star RA Illei GG Alevizos Exosomes from human saliva as a source of microRNA biomarkers Oral Dis 2010 Jan 16 1 34 8 Luo SS Ishibashi O Ishikawa G Ishikawa T Katayama A Mishima T Takizawa T Shigihara T Goto T Izumi A Ohkuchi
11. nute exoRNA check to see that all flowed through Purification otherwise spin longer 13 Discard flow through and place spin column back into collection tube 14 Add 400ul WASH Buffer 888 266 5066 Toll Free 650 968 2200 outside US Page 3 System Biosciences SBI User Manual 15 Centrifuge at 13 000 rpm for 1 minute 16 Repeat steps 13 to 15 once again total of 2 Washes 17 Discard flow through and centrifuge at 13 000 rpm for 2 minutes to dry IMPORTANT Discard collection tube and assemble exoRNA spin column with a fresh Elution RNase free 1 5ml elution tube not provided Add 30ul ELUTION Buffer directly to membrane in spin column Centrifuge at 2 000 rpm for 2 minutes loads buffer in membrane Increase speed to 13 000 rpm and centrifuge for 1 minute elutes exoRNAs You should have recovered 30 40ul exosome RNA The yield of RNA from isolated exosomes is different depending on the starting biofluid or the type of cells that were grown in culture Different cell types secrete varying levels of exosomes For serum the level of RNA isolated from 500 ul is usually in the 500ng range and can be measured using a Agilent Bioanalyzer or a NanoDrop Spectrophotometer B Using Precipitated Exosomes for Protein Extraction ELISA analysis SBI offers three ELISA kits Catalog ExoELISA 63 ExoELISA 9 ExoELISA 81 for fast and quantitative analysis of well characterized exosomal protein markers CD63 CD9 and CD81
12. ormed to analyze the presence of the specific marker protein placental alkaline phosphatase PLAP The SDS PAGE gel was transferred to a nitrocellulose membrane the membrane blocked for 1 hour at room temperature with non fat dried milk and probed overnight at 4 C with primary antibody The bound immune complexes were visualized by enhanced chemiluminescence ECL Amersham Life Sciences and quantitated by densitometry Un Scan it Software Silk Scientific Corp MicroRNA Yield from Exosomes precipitated with ExoQuick versus other Extraction Methods Fug g 30 Small RNA 16 660 5 pg ul U s fmicroRNA 8 904 1 pg nl we Cian Ratio 53 Q 15 os 10 5 0 25 30 35 40 45 50 55 60 65 is ru Small RNA 5 297 3 pg ul I w sof imicroRNA 1 119 5 pg ul H s Ratio 21 A N i Cd Pra 6 r d 10 O i ismall RNA 11 116 0 pg ul microRNA 5 255 4 pg ul Ratio 47 DynaBeads 25 30 35 40 45 50 55 60 65 Is Agilent Bioanalyzer data courtesy of Dr Douglas Taylor Univ Lousiville KY Page 6 ver 4 2011 07 11 www systembio com ExoQuick Exosome Precipitation Solution Cat EXOQ_ A 1 The RNA quality and yield was accessed using a GeneQuant Il Small RNAs were analyzed with the Agilent 2100 Bioanalyzer Lab on a Chip instrument system Agilent Technologies using the Agilent Small RNA chip and reagent kit Approximately 100ng of isolated total RNA in 1ul was applied to each run The manufacturer s recommended pro
13. reagent rapidly into plasma sample to mix thoroughly 3 Incubate at 37 C for 15 minutes 4 Spin at 10 000 rpm at RT for 5 minutes microfuge 5 Save supernatant serum like sample discard coagulated pellet 6 Use serum like supernatant with ExoQuick to isolate exosomes as stated in ExoQuick protocol use 25ul ExoQuick per 100ul coagulated plasma supernatant References As featured in Exosome Isolation for Proteomic Analyses and RNA Profiling Douglas D Taylor Wolfgang Zacharias and Cicek Gercel Taylor Serum Plasma Proteomics Methods in Molecular Biology 2011 Volume 728 Part 4 235 246 PDF Tae Hoon Lee Esterina D Asti Nathalie Magnus Khalid Al Nedawi Brian Meehan and Janusz Rak Review Microvesicles as mediators of intercellular communication in cancer the emerging science of cellular debris Seminars in Immunopathology DOI 10 1007 s00281 011 0250 3 PDF Technical References Adachi T Nakanishi M Otsuka Y Nishimura K Hirokawa G Goto Y Nonogi H Iwai N Plasma microRNA 499 as a biomarker of acute myocardial infarction Clin Chem 2010 Jul 56 7 1183 5 De Smaele E Ferretti E Gulino A MicroRNAs as biomarkers for CNS cancer and other disorders Brain Res 2010 Jun 18 1338 100 11 Mitchell PS Parkin RK Kroh EM Fritz BR Wyman SK Pogosova Agadjanyan EL Peterson A Noteboom J O Briant KC Allen A Lin DW Urban N Drescher CW Knudsen BS Stirewalt DL Gentleman R Vessella RL Nelso
14. tibodies against two commonly found exosome biomarker proteins CD63 CD9 CD81 and Hsp70 and small RNAs analysed on RNA gels after Trizol extraction Exosome Protein Marker Analysis ExoAbs gt a ae J LEL P PS x gt va kDa SP SS Pe woo MWe 70 55 35 25 ExoQuich Excsome Serum Western Analysis Exosome RNA Analysis 4 15 PA RNA Gel www systembio com ExoQuick Exosome Precipitation Solution Cat EXOQ_ A 1 ll Exosome Precipitation Protocol Isolate exosomes with ExoQuick 1 Collect biofluid and centrifuge at 3000 x g for 15 minutes to remove cells and cell debris 2 Transfer supernatant to a sterile vessel and add the appropriate volume of ExoQuick Exosome Precipitation Solution to the bio fluid Some examples are shown in the Table below Mix well by inverting or flicking the tube Note when precipitating exosomes from plasma the resulting pellet may be difficult to resuspend due to Incubation Sample ExoQuick precipitated fibrin and other fibrinogens For plasma please refer to the Appendix for additional preparation steps Time Bio fluid volume volume 30 minutes Serum 250 ul 63 ul Overnight Ascites fluid 250 ul 63 ul Refrigerate overnight at least 12 hours The tubes do not need to be rotated during the incubation period 4 Centrifuge ExoQuick biofluid mixture at 1500 x g for 30 minutes Centrifugation may be performed at either room temperature or 4 C with similar
15. tocol was strictly followed to obtain Bioanalyzer profiles for the size range 6 to 150 nucleotides nt The profiles were calibrated for size nt using the small RNA ladder supplied with the kit containing markers of 20 40 60 80 and 150 nt in size as reference The instrument software quantitated the peak area between 0 and 150 nt as small RNA region the area within 10 to 40 nt as microRNA region and provides percentages of miRNA detected for each sample 888 266 5066 Toll Free 650 968 2200 outside US Page 7 System Biosciences SBI User Manual 4 Activity Assays Track Exosomes using Cyto Tracers SBI has created a line of lentivector based Cyto Tracers that utilize GFP fusion proteins to mark cellular compartments organelles vesicles and structures to enable more long term and more in depth experimentation The Cyto Tracers can be used in transfections as well as packaged into virus to create stable GFP tracer cell lines in primary cells tumor cell lines and stem cells The Tetraspanin CD63 protein is a common biomarker for exosomes With the pCT CD63 GFP construct you can make you cells of interest secrete exosomes that glow green for downstream functional delivery studies Cat CYTO120 PA 1 Transduced 293 cells pCT CD63 GFP CD63 Tetraspanin Tag Catalog CYTO120 PA 1 gt Transfected 293 cells lt 1 NanoSight The NanoSight LM10 instrument is based on a conventional optical microscope and
16. uses a laser light source to illuminate nano scale particles within a 0 3 ml sample introduced to the viewing unit with a disposable syringe Enhanced by a near perfect black background particles appear individually as point scatterers moving under Brownian motion The image analysis Nanoparticle Tracking Analysis NTA software suite allows users to automatically track and size nanoparticles on an individual basis Results are displayed as a frequency size distribution graph and output to spreadsheet ExoQuick serum exosome analysis Normal human serum from 50 pooled samples was used Only 250ul serum was combined with 63ul ExoQuick to pellet the exosomes in 30 minutes The exosome pellet was resuspended in 100ul PBS diluted 1 10 000 and visualized on the NanoSight LM10 instrument The analysis shows that ExoQuick isolated 90nm exosomes with a recovery of 2 74 x 10 12 particles ml Page 8 ver 4 2011 07 11 www systembio com V Vi ExoQuick Exosome Precipitation Solution Cat EXOQ_ A 1 NANOSCHT i ExoQuick isolates I 90nm exosomes from serum 0 100 200 300 400 500 600 700 800 900 m Particle Size nm Data acquisition and analysis These experiments were done in collaboration with Particle Characterization Laboratories Inc PCL Appendix Pacific Hemostasis Thromboplastin D Thermo cat 10 0356 1 Use amount of Thromboplastin D in Pacific Hemostasis protocol 2 Squirt in Thromboplastin D
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