Isolation. mircury Exosome Isolation Kit Serum and plasma

Transcription

1 Isolation mircury Exosome Isolation Kit Serum and plasma Instruction manual v1.2 # October 2015

2 Table of Contents Product summary mircury Exosome Isolation Kit Serum and plasma Additional required material Product description Storage and product stability Protocol & notes Exosome isolation from Serum /plasma Notes prior to use Section A. Exosome isolation from 1.4 ml serum Section B. Exosome isolation from 0.5 ml serum Section C. Exosome isolation from 1.4 ml plasma Section D. Exosome isolation from 0.5 ml plasma Tips and troubleshooting Related products Appendix

3 Product summary mircury Exosome Isolation Kit Serum and plasma The mircury Exosome Isolation Kit Serum and plasma consists of the components described in Table 1. Table 1. Kit Components (16-50 isolations) Amount supplied Precipitation Buffer A 10 ml Resuspension Buffer 10 ml Thrombin (lyophilized)* 200 U Thrombin Buffer* 500 µl * *Preparation of working solution required see notes prior to use. Additional required material For All Protocols Benchtop microcentrifuge Pipette (+ RNase free tips) Vortexer / multi-vial vortex shaker Refrigerator 1.5 or 2 to 2.2 ml vials depending on the sample volume used mircury RNA Isolation Kit Biofluids (for downstream RNA isolation) 3

4 Product description Exiqon s mircury Exosome Kit Serum and plasma provides a rapid method for the isolation and purification of exosomes from serum and plasma Exosomes are cell derived membranous particles with a size of 20 to 120 nm, approximately the same size as viruses but considerably smaller than microvesicles. Exosomes are excreted from cells into the surrounding media and can be found in many if not all body fluids. Their proposed role as intercellular hormone like messenger together with their stability as carrier of proteins and RNA makes them ideal in the search for biomarkers for a variety of biological questions. The isolation is based on capturing of water molecules which otherwise form the hydrate envelope of particles in suspension. By mixing the sample with the Precipitation Buffer the hydration of the particles will be diminished. This allows precipitation of the subcellular particles below 100 nm with a low speed centrifugation step. The protocols cover different sample volumes of serum and plasma. They are validated to allow subsequent microrna isolation using the mircury RNA Isolation Kit - Biofluids and improve the quality of the obtained microrna signature according to the exosomal subpopulation analyzed. Furthermore increased amount of sample can be used with little effect on downstream PCR analysis from inhibitors. The fractionation is started by pelleting of dead cells, debris, platelets or similar. A more complete removal of platelets or large microvesicles can be obtained starting with a simple filtration of the sample through 0.2 µm filter spin columns (not provided). Such a step has been shown beneficial for certain biofluid biomarker signatures (Cheng et al.,doi: / journal.pone ). The protocols consist of 5 simple steps (see also Figure 1): 1. Thawing samples on ice or at 4 C 2. Pelleting of dead cells / debris / platelets / fibrin 3. Mixing of the sample with the Precipitation Buffer and incubation at 4 C 4. Pelleting of the exosomal fraction 5. Resuspension for further processing or characterization 4

5 Figure 1. Protocol overview of the mircury Exosome Isolation Kit Serum and plasma, followed by RNA isolation using the mircury RNA Isolation Kit Biofluids (prod no ). Spin the sample to remove dead cells and debris Mix sample with Precipitation Buffer incubate 60 min at 4 C Spin discard supernatant & resuspend pellet Take resuspended pellet as starting material for mircury RNA Isolation Kit Biofluids Important note - cautions Ensure that a suitable lab coat, disposable gloves and protective goggles are worn and standard safety precautions are followed when working with chemicals. For more information, please consult the appropriate Material Safety Data Sheets (MSDSs). Body fluids like serum or plasma of all human and animal subjects are considered potentially infectious. All necessary precautions recommended by the appropriate authorities in the country of use should be taken when working with body fluids. 5

6 Table 2 Kit Specifications Particles precipitated Exosomes, microvesicles and to some degree larger protein complexes Maximum volume of starting material supported 1400 µl Recommended volume of starting material µl Resuspension volume 300 µl Time to complete 10 isolations <120 min Storage and product stability All solutions should be kept tightly sealed and stored protected from light at 2-8 C. These reagents should remain stable for at least 6 months in their unopened containers. Thrombin is shipped lyophilized at room temperature and should be stored at 2-8 C upon arrival. Thrombin will be stable for at least 6 month after resuspension when stored at 2-8 C. Important note - Transportation/storage of biofluid samples When storing and transporting biofluid samples intended for exosome isolation, it is recommended to use 4 C or -20 C. If samples are frozen they should be centrifuged prior to freezing. Store or ship the cell free supernatant to avoid contamination of the samples with cellular components that otherwise may be released from cells during freeze thawing. 6

7 Protocol & notes Exosome isolation from Serum / plasma Notes Prior to Use Make sure that the sample collection and treatment and storage up to this point has been as uniform as possible amongst the individual samples. A ny preparation is highly dependent on the amount of starting material. The standard protocol is flexible to extract exosomes from 0.2 ml up to 1.4 ml starting material without any additional steps simply by adjusting the amounts of Precipitation Buffer. The exosome composition from serum and plasma may give different results in downstream analysis depending on the treatment of the body fluid prior to RNA isolation. Please ensure that sample acquisition conditions and specimen pretreatment are controlled and defined, e.g. if interested in a cell free specimen with low platelet content, centrifuge with 3,000 x g (~2,000 RPM) for 5 to 10 minutes in order to pellet cells, debris and platelets. Transfer the supernatant as fraction of interest into a new vial prior to use or storage. For downstream RT-qPCR we recommend not to work with hemolysed samples. Even traces of red blood cells in the serum or plasma will affect the microrna profile. It is recommended to follow the microrna QC PCR manual: For downstream RT-qPCR we recommend the use of serum or citrate/edta plasma and discourage using heparin plasma. RNA isolated from heparin plasma can reduce PCR performance. Important note Prepare thrombin working solution before proceeding to manual Prepare a working solution of thrombin by adding 400 µl Thrombin Buffer to the Thrombin vial. Incubate at room temperature for 1 min. Swirl gently to redissolve completely but avoid vigorous mixing to minimize mechanical shearing which could affect enzyme quality. Aliquot and store at -20 C for later use. Avoid repeated freeze thawing. The working solution is stable for at least 6 months. 7

8 Four protocols are available depending on starting volume and sample type: The protocols are scalable and can be used for any starting volume from 0.2 ml to 1.4 ml depending on the need of the downstream purpose. For qpcr we recommend the use of 0.5 to 1.0 ml sample material as starting volume. Section A. Protocol for exosome isolation from 1.4 ml serum, please go to page 9 Section B. Protocol for exosome isolation from 0.5 ml serum, please go to page 10 Section C. Protocol for exosome isolation from 1.4 ml plasma, please go to page 11 Section D. Protocol for exosome isolation from 0.5 ml plasma, please go to page 12 Downstream RNA isolation If extracting RNA from the isolated exosomes, please use mircury RNA Isolation Kit Biofluids for optimal result. RNA isolation protocol found in appendix page 16. Any RNA spikeins should be added in the RNA isolation procedure (see page 16) to prevent RNA degradation of the spike-ins. 8

9 Section A. Exosome isolation from 1.4 ml serum Notes prior to use Before getting started please ensure that the centrifuge is run at room temperature. Thaw samples on ice or at 4 C. Vortex Precipitation Buffer prior to use. Step 1 Spin 1.5 ml serum for 5 minutes at 10,000 x g to remove cell debris. Step 2 Transfer 1.4 ml of supernatant into a new 2 ml reaction vial. Step 3 Add 560 µl Precipitation Buffer A and vortex for 5 seconds to mix. Step 4 5 Incubate for 60 minutes at 4 C.* Step 5 Incubate Spin for 30 forminutes 60 minutes at 1,500 at 4 C.* x g at room temperature. Step 6 Remove supernatant completely and discard (or save for separate analysis).briefly respin to collect and remove residual supernatant Step 7 Resuspend pellet by vortexing in 240 µl Resuspension Buffer ending up with ~300 µl final volume. For multiple samples use a 2 ml reaction vial vortex shaker for 5 to 15 minutes at room temperature. Step 8a Continue RNA extraction with the resuspended pellet using mircury RNA Isolation Kit Biofluids (use protocol from appendix in this manual).. Step 8b Storage The purified exosome sample may be stored at 4 C for up to 2 days or can be stored at -20 C prior to RNA isolation. In order to minimize the risk of RNase contamination we recommend proceeding directly with further downstream sample processing. * Precipitation time can be extended e.g. overnight. 9

10 Section B. Exosome isolation from 0.5 ml serum Notes prior to use Before getting started please ensure that the centrifuge is run at room temperature. Thaw samples on ice or at 4 C. Vortex Precipitation Buffer prior to use. Step 1 Spin 0.6 ml serum for 5 minutes at 10,000 x g to remove cell debris. Step 2 Transfer 0.5 ml of supernatant into a new 1.5 ml reaction vial. Step 3 Add 200 µl Precipitation Buffer A and vortex for 5 seconds to mix. Step 4 5 Incubate for 60 minutes at 4 C.* Step 5 Incubate Spin for 30 forminutes 60 minutes at 1,500 at 4 C.* x g at room temperature. Step 6 Remove supernatant completely and discard (or save for separate analysis). Briefly respin to collect and remove residual supernatant. Step 7 Resuspend pellet by vortexing in 270 µl Resuspension Buffer ending up with ~300 µl final volume. For multiple samples use a reaction vial vortex shaker for 5 to 15 minutes at room temperature. Step 8a Continue RNA extraction with the resuspended pellet using mircury RNA Isolation Kit Biofluids (use protocol from appendix in this manual). Step 8b Storage The purified exosome sample may be stored at 4 C for up to 2 days or can be stored at -20 C prior to RNA isolation. In order to minimize the risk of RNase contamination we recommend proceeding directly with further downstream sample processing. * Precipitation time can be extended e.g. overnight. 10

11 Section C. Exosome isolation from 1.4 ml plasma Notes prior to use Before getting started please ensure that the centrifuge is run at room temperature and that the Thrombin has been resuspended in the Thrombin Buffer. Thaw samples on ice or at 4 C. Vortex Precipitation Buffer prior to use. Step 1 Add 17 µl Thrombin (stock concentration of 500 U/mL) to 1.7 ml plasma, in a 2.0 ml reaction vial. Mix and incubate for 5 minutes at room temperature. Step 2 Spin for 5 minutes at 10,000 x g. Step 3 Transfer 1.4 ml of supernatant into a new 2.0 ml reaction vial. Step 4 Add 560 µl Precipitation Buffer A and vortex for 5 seconds to mix. Step 5 Incubate for 60 minutes at 4 C.* Step 6 Spin for 5 minutes at 500 x g at room temperature. Step 7 Remove supernatant completely and discard (or save for separate analysis). Briefly respin to collect and remove residual supernatant. Step 8 Resuspend pellet by vortexing in 240 µl Resuspension Buffer ending up with ~300 µl final volume. For multiple samples use a 2 ml reaction vial vortex shaker for 5 to 15 minutes at room temperature. Step 9a For RNA extraction continue with the resuspended pellet using mircury RNA Isolation Kit Biofluids (use protocol from appendix in this manual). Step 9b Storage The purified exosome sample may be stored at 4 C for up to 2 days or can be stored at -20 C prior to RNA isolation. In order to minimize the risk of RNase contamination we recommend proceeding directly with further downstream sample processing. * Precipitation time can be extended e.g. overnight. 11

12 Section D. Exosome isolation from 0.5 ml plasma Notes prior to use Before getting started please ensure that the centrifuge is run at room temperature and that the Thrombin has been resuspended in the Thrombin Buffer. Thaw samples on ice or at 4 C. Vortex Precipitation Buffer prior to use. Step 1 Add 6 µl Thrombin (stock concentration of 500 U/mL) to 0.6 ml plasma, mix and incubate for 5 minutes at room temperature. Step 2 Spin for 5 minutes at 10,000 x g. Step 3 Transfer 0.5 ml of supernatant into a new 1.5 ml reaction vial. Step 4 Add 200 µl Precipitation Buffer A and vortex for 5 seconds to mix. Step 5 Incubate for 60 minutes at 4 C.* Step 6 Spin for 5 minutes at 500 x g at room temperature. Step 7 Remove supernatant completely and discard (or save for separate analysis). Briefly respin to collect and remove residual supernatant. Step 8 Resuspend pellet by vortexing in 270 µl Resuspension Buffer ending up with ~300 µl final volume. For multiple samples use a 2 ml reaction vial vortex shaker for 5 to 15 minutes at room temperature. Step 9a Continue RNA extraction with the resuspended pellet using mircury RNA Isolation Kit Biofluids (use protocol from appendix in this manual). Step 9b Storage The purified exosome sample may be stored at 4 C for up to 2 days or can be stored at -20 C prior to RNA isolation. In order to minimize the risk of RNase contamination we recommend proceeding directly with further downstream sample processing. * Precipitation time can be extended e.g. overnight. 12

13 Tips and Trouble shooting No pellet visible Serum and plasma should give relatively large pellets. In case of an absence of any visible pellet, please check that the Precipitation Buffer has been added and that the incubation at 4 C has been carried out for at least 60 min. Pellet not completely resuspended If the plasma sample has been spun with more than 500 x g, the pellet will be hard to resuspend. We have observed good RNA isolation from the partially resuspended pellet after addition of the mircury RNA Isolation Kit - Biofluids lysis buffer. Work swiftly when proceeding from step 7. Do not allow the pellet to stand longer than necessary. Platelet/microvesicle removal Pretreatment of serum and plasma samples should be uniform. Platelets should be separated from plasma with initial density centrifugation after sample acquisition in order to obtain platelet poor plasma. In addition potentially present microvesicles (and platelets) can be removed by a filtration step through a 0.2 µm µm filter (not provided) before the actual precipitation step. Low RNA yield from exosomes If the intended downstream application requires higher RNA concentration, consider reducing the recommended elution volume for exosome derived RNA of 100 µl in the mircury RNA Isolation Kit Biofluids to the standard 50 µl. 13

14 Related products Exiqon offers a specialized RNA isolation kit enabling preparation from larger amounts of human/animal tissues as well as cells and plants and downstream application products to analyze and verify the expression, function and spatial distribution of micrornas: mircury RNA Isolation Kit Biofluids Small RNA preparations from serum or plasma samples or from exosomes from serum or plasma. Suitable for mircury LNA Universal RT microrna PCR. mircury RNA Isolation kit Cell & Plant Total RNA preparations from cultured animal cells, small tissue samples, blood, bacteria, yeast, fungi, bacteria, plants or exosomes from cells, urine, CSF or similar. mircury RNA Isolation kit -Tissue Get high quality total RNA suitable for mircury LNA Universal RT microrna PCR or mircury LNA microrna Array analysis in as little as 20 minutes. Total RNA preparations from mg animal/human tissue. mircury LNA Universal RT microrna PCR Exiqon s microrna qpcr system offers the best available combination of performance and ease-of-use on the microrna real-time PCR market. The combination of a Universal RT reaction and LNA -enhanced PCR primers results in unmatched sensitivity and specificity. The Ready-to-use microrna PCR panels enable fast and easy microrna expression profiling. Take advantage of the tailored Universal RT microrna PCR spike in kit to monitor the performance of your PCR. mircury LNA microrna Array, microarray kit Pre-printed mircury LNA microrna Array microarray slides, available in pack sizes of 3, 6 and 24 for hsa, mmu & rno and other species. The kit comes complete with hybridization and wash buffers as well as synthetic spike-in micrornas. mircury LNA microrna Detection For in situ hybridization and northern blotting of all annotated micrornas. mircury LNA microrna ISH Buffer Set (FFPE). 14

15 mircury LNA microrna ISH Optimization kit (FFPE) Complete kit with control probes and hybridization buffer for easy set up of microrna in situ hybridization. mircury LNA microrna Inhibitors and Power Inhibitors Unravel the function of micrornas by microrna inhibition. Sophisticated LNA design ensures potent inhibition of all micrornas regardless of their GC content. Chemically modified, highly stable Power Inhibitors for unrivaled potency. mircury LNA microrna Inhibitor Library For genome-wide high throughput screening of microrna function. 15

16 Appendix RNA preparation from exosomes (modified from mircury RNA Isolation Kit Biofluids). Notes prior to use Before getting started please ensure that isopropanol is available and that ethanol has been added to Wash Solution 2 BF. Make sure that the centrifuge is run at room temperature. Step 1 Lysis Mix 300 µl supernatant from exosome isolation with 90 µl Lysis solution BF Vortex for 5 sec. Incubate for 10 min at room temperature. Recommendation: For downstream PCR analysis, add 1 µl RNA Spike-in template mixture (mircury LNA Universal RT microrna PCR, RNA Spike-in kit) and 1µg carrier-rna/90µl Lysis Solution BF. 16 Step 2 Protein Precipitation Add 30 μl of Protein Precipitation Solution BF. Vortex for 5 sec. Incubate for 1 min at room temperature. Centrifuge for 3 min at 11,000 x g. Step 3 Transfer supernatant Transfer the clear supernatant into a new collection tube (2 ml, with lid). Step 4 Adjust binding conditions Add 400 µl Isopropanol. Vortex for 5 sec. Step 5 Load column Place a microrna Mini Spin Column BF in a collection tube and load sample onto the column. Incubate for 2 min at room temperature. Centrifuge for 30 sec at 11,000 x g. Discard flow-through and place column back into the collection tube.

17 Step 6a Wash and dry Add 100 µl Wash Solution 1 BF to the microrna spin column BF. Centrifuge for 30 sec at 11,000 x g. Discard flow-through and place column back into the collection tube. Step 6b Wash and dry Add 700 µl Wash Solution 2 BF to the microrna spin column BF. Centrifuge for 30 sec at 11,000 x g. Discard flow-through and place column back into the collection tube. Step 6c Wash and dry Add 250 µl Wash Solution 2 BF to the microrna spin column BF. Centrifuge for 2 min at 11,000 x g to dry the membrane completely. Step 7 Elute Place the microrna spin column BF in a new collection tube (1.5 ml). Add 100 µl RNase free H2O directly onto the membrane of the microrna spin column BF. Incubate for 1 min at room temperature. Close the lid and centrifuge for 1 min at 11,000 x g. Step 8 Storage The purified RNA sample may be stored at 20 C for a few days. It is recommended to keep the samples at 70 C for long term storage. Note: For downstream qpcr analysis we recommend using the PCR manual for serum/plasma and other biofluid samples, using the recommended cdna input from Table 3. 17

18 Literature citations: Please refer to mircury Exosome isolation kit Serum and plasma when describing a procedure for publication using this product. Patents and Trademarks Exiqon and mircury are registered trademarks of Exiqon A/S, Vedbaek, Denmark. Cautions and Disclaimer Products are for research use only and not for diagnostic or therapeutic use. The products may be used only for the buyer s internal research purposes and not for commercial use. The buyer may not resell products in their original or any modified form. The purchase of products does not include or carry an implied right or license for the buyer to use such products in the provision of services to third parties and a license must be obtained directly from Exiqon A/S for such use. Ensure that a suitable lab coat, disposable gloves and protective goggles are worn when working with chemicals. For more information, please consult the appropriate Material Safety Data Sheets (MSDSs). Body fluids of all human and animal subjects are considered potentially infectious. All necessary precautions recommended by the appropriate authorities in the country of use should be taken when working with body fluids. Copyright 2014 Exiqon. All rights reserved 18

19 Notes 19

20 Outside North America Exiqon A/S Skelstedet 16 DK-2950 Vedbaek Denmark Phone Fax North America Exiqon Inc. 12 Gill Street, Suite 1650 Woburn, MA United States Phone (781) Fax (781) v1.2-10/2015 exiqon.com