ExoQuick-LP User manual
Contents
1. SSBI System Biosciences ExoQuick LP Exosome Isolation Kit Cat EXOLPxA 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 contained in this user manual ExoQuick LP Cat EXOLP5A 1 Contents I Overview Caaa 2 ll ExoQuick LP Protocol ss 4 ll Sample Data for ExoQuick LP sceeeeeeeeees 5 IV PICICLONGCCS arene a 8 V Technical SUD POM esirinnas 8 VI Licensing ANd Walrranty ccccsceeeeeeneeeeeeeeeeeeeneeeens 8 List of Components in ExoQuick LP Cat EXOLP5A 1 Item Volume ExoQuick exosome precipitation solution 1ml pm Pre Clearing Reagent A 25 ul Pre Clearing Reagent B 25 ul Magnetic Bead Solution 500 ul Magnetic stand for pulling beads is not included in the kit but can be purchased separately from SBI Cat EXOFLOW700A 1 The ExoQuick LP kit components are shipped on blue ice and should be stored at 4 C upon receipt Pre Clearing Reagent A and B can be stored at 4 C upon receipt for 1 month For long term use please store Pre Clearing Reagent A and B at 20 C Properly stored kits are stable for 1 year from the date received The kit provides enough ExoQuick 1 mL for processing up to 500 ul of serum or plasma samples good for 5 reactions with each reaction defined as a single 100 ul s2. to efficiently deplete lipoprotein particles from plasma or serum before ExoQuick precipitation Page 2 ver 1 2013 07 15 www systembio com ExoQuick LP Cat EXOLP5A 1 11 12 13 14 19 16 17 ExoQuick LP protocol Use two 1 5 ml microcentrifuge tubes for one serum sample 100 ul If starting material is plasma it must be pre treated with SBI s Thromboplastin D reagent please see protocol here http Awww systembio com downloads PAC EXOQ5TD 1 pdf prior to starting the ExoQuick LP protocol Transfer 50 ul of magnetic bead suspension into each of the 1 5ml microcentrifuge tubes and add 300 ul of wash buffer to each tube and vortex briefly Place the tubes on a 1 5ml microcentrifuge tube magnetic separator and discard the supernatant after beads are collected Repeat wash step with 300 ul of wash buffer Centrifuge tubes briefly resuspend beads in 100 ul of wash buffer in each 1 5ml microcentrifuge tube Add 5 ul of Pre Clearing Reagent A to one tube and 5 ul Pre Clearing Reagent B to another tube respectively Incubate the two tubes with rotation for 30 60 minutes at room temperature Centrifuge the tubes briefly place on a magnetic separator and pipette off the supernatant Repeat the wash step Remove tubes from the magnetic separator add 200 ul of wash buffer to each tube and combine beads into one tube Centrifuge 0 5 ml of human serum sample at 3000 x g for 15 minutes to remove cells and cell debris
3. you should have any questions or concerns about any SBI products please contact us at 888 266 5066 2013 System Biosciences SBI All Rights Reserved Page 6 ver 1 2013 07 15 www systembio com
4. BS buffer and then diluted 1 2 000 before sending to Particle Characterization Lab for visualization and analysis The results are shown as a plot of frequency vs particle size Fig 3 The data indicates that there are minimal differences in particle sizes using ExoQuick LP vs untreated controls suggesting that exosomes are structurally intact Page 4 ver 1 2013 07 15 www systembio com ExoQuick LP Cat EXOLP5A 1 NonFilter pre treatment x i NonFilter ExoQuick direct i 3 Filter pre treatment i A carer re Filter ExoQuick direct _ _ uae X m Figure 3 NanoSight analysis plots of frequency vs particle size in unfiltered and filtered samples that were untreated or ExoQuick LP treated IV References Vickers KC and Remaley AT Lipid based carriers of microRNAs and intercellular communication Curr Opin Lipidol 2012 Apr 23 2 91 7 Vickers KC Palmisano BT Shoucri BM Shamburek RD Remaley AT MicroRNAs are transported in plasma and delivered to recipient cells by high density lipoproteins Nat Cell Biol 2011 Apr 13 4 423 33 V Technical Support For more information about SBI products and to download manuals in PDF format please visit our web site http www systembio com For additional information or technical assistance please call or email us at 888 266 5066 Toll Free 650 968 2200 outside US Page 5 System Biosciences SBI Use
5. ae 4 Simple one step i precipitation 35 25 ExoQuick Exosoene Serem Western Analysis Exosome RNA Analysis amp Supernatant S Exosome pei pellet 15 PA RNA Gel Human blood plasma contains a heterogeneous mixture of microvesicles exosomes apoptotic bodies and lipoprotein particles These particles have been previously shown to carry nucleic acid cargo in the form of microRNAs Vickers et al 2012 Therefore circulating microRNAs are a mixture of microRNAs present in the aforementioned subcellular particles These microRNAs are known to be quite stable in blood plasma and the levels of specific circulating microRNAs can differ with diseases leading to the possibility of using extracellular microRNAs as disease biomarkers In order to study only the exosome associated microRNAs and proteins it is important to remove as many subcellular particles as possible from blood plasma or serum before the treatment with ExoQuick particularly lipoprotein particles Lipoproteins are the major lipid based particles in the plasma and serum It has been recently demonstrated that both high density lipoproteins HDLs and low density density lipoproteins LDLs contain microRNAs Vickers et al 2011 In order to deplete as much of these lipoproteins which may cause false positive hits in analysis of exosome associated microRNAs we have developed the ExoQuick LP kit containing ExoQuick and proprietory pre clearing reagents
6. ample If starting material is plasma it must be pre treated with SBI s Thromboplastin D reagent please see protocol here http www systembio com downloads PAC EXOQ5TD 1 pdf prior to starting the ExoQuick LP protocol 888 266 5066 Toll Free 650 968 2200 outside US Page 1 System Biosciences SBI User Manual 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 and accurately profile them using SBI s SeraMir gPCR 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 or bio fluid H Exosome precipitation from Serum or ExoQuick 250 ul human serum samples Biofluid Solution was verified by Western blot using antibodies against two commonly found exosome biomarker proteins CD63 CD9 CD81 and Hsp70 and small RNAs analyzed on RNA gels l after Trizol extraction SSB Exosome Protein Marker Analysis gt a vw 3 Ne kDa S Le amp 100 er
7. and collect the supernatant Pass the above mentioned supernatant through a 0 22 um filter and collect the serum Add 100 ul of filtered serum into the bead mixture Incubate the tube with rotation for 3 hours at 4 C After incubation centrifuge the tube briefly and place on a magnetic separator Transfer the supernatant into a new tube which contains lipoprotein depleted serum Add ExoQuick using 1 5 of the volume of the supernatant into the tube containing supernatant and mix well by inverting the tube or shaking the tube up and down Incubate at 4 C overnight Centrifuge the tube at 14000 rpm for 10 minutes next day Aspirate the supernatant and the remaining pellet contains lipoprotein depleted exosomes for downstream applications Please refer to the full ExoQuick user manual http www systembio com downloads Manual_ExoQuick_WEB pdf for protocol suggestions on isolating protein and RNA from the pellet Sample Data for ExoQuick LP ApoA1 is the major protein component of high density lipoprotein or HDL comprising approximately 70 of the protein moiety of HDL ApoE is a protein found mainly in low density lipoprotein or LDL ApoE is also found with a smaller amount in HDL 888 266 5066 Toll Free 650 968 2200 outside US Page 3 System Biosciences SBI User Manual Treatment of serum samples with Pre Clearing Reagents A and B significantly reduces the amount of both ApoA1 and ApoE in ExoQuick serum pellet as me
8. asured by ELISA Fig 1 ApoA 1 ApoE K RN N ee lt Figure 1 Validation data showing treatment of serum samples with SBI s ExoQuick LP reagents leads to significant reduction of ApoA1 and ApoE lipoproteins We used Exosome ELISA kits SBI Cat EXOEL CD81A 1 EXOEL CD63A 1 and EXOEL CD9A 1 to measure the approximate number of exosomal particles present in ExoQuick serum pellets with or without treatment with ExoQuick LP Pre Clearing Reagents Fig 2 While there were some differences in observed particle numbers in untreated vs treated samples overall yields were quite similar suggesting minimal loss of material using ExoQuick LP 3 00 2 50 E treat with Pre clean reagents 2 00 E No pre treatment 1 50 Exosome particles 1x10 8 1 00 0 50 O O 0 00 CD81 CD63 9 Figure 2 Validation data showing overall exosome particle numbers comparing ExoQuick pellets that were treated with ExoQuick LP vs untreated control To visually test for exosome integrity we performed dynamic light scattering analysis of untreated and ExoQuick LP treated exosomes using the NanoSight LM10 instrumentation see previous section for details In brief we passed 100 ul of serum through a 0 22 um filter before treating with ExoQuick LP as well as an untreated control We also collected samples that were not filtered through a 0 22 um filter for comparison The ExoQuick serum pellets were resuspended in 100 ul P
9. icense 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 will 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
10. r Manual 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 VI Licensing and Warranty Statement Limited Use License Use of the ExoQuick Exosome Precipitation Solution 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 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 l
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