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TotalExoRNA™ Overall exosome immunocapture and RNA extraction kit (Biological Fluids/Cell Media, 10 reactions)

Details for Product No. ABIN2868142, Supplier: Log in to see
Application
Immunoassay (IA), RNA Extraction (REx)
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Purpose TotalExoRNA™ Overall exosome immunocapture and RNA extraction kit from biofluids and cell media
Brand TotalExoRNA™
Sample Type Urine, Tissue Culture Medium, Cell Culture Cells, Biological Fluids
Characteristics TotalExoRNA™ Kit (overall exosome immunocapture and RNA extraction kit) has been developed for the isolation and efficient extraction of high-quality total RNA (miRNA and mRNAs) from the overall exosome population from human biofluids or cell culture media. Exosome capture occurs on immunobeads precoated with exosome associated antibodies enabling overall exosome isolation. The immunobeads can capture the overall exosome population from a wide range of media, including cell culture supernatants and human biofluids (plasma, serum, urine, etc) as well as enrich some specific exosome subpopulations (tumor-derived exosomes). The captured exosomes are subsequently lysed with an optimized lysis buffer and total RNA is purified using spin columns with a fast and user-friendly process. Eluted RNA can be used for downstream analyses or stored at -80 °C. Exosome standards for positive control are also included in the kit. All our kits guarantee high specificity for exosomal RNA and high yield of total RNA (including small RNAs) than similar products.
Components Pre-couple latex immunobeads, Bead Washing buffer (5X), Lysis buffer, RNA Washing buffer (to add Ethanol 96%), Elution buffer, Columns, RNase free elution tubes (1.5 ml), Lyophilized Exosome Standard
Material not included
  • Single-use and/or pipettes with disposable tips 2-100 μL
  • Pipettes 1 mL and 5 mL for reagent preparation
  • PBS
  • Disposable pipetting reservoirs
  • Ethanol 96 %
  • Chloroform or BCP (1-bromo-3-chloropropane)
  • Sample concentrator (urine and cell culture supernatant samples)
Background Overall Exosome Immunocapture and RNA extraction Kit (from human biofluids and cell media)
Comment

Further details regarding sample type: Human biological fluids including Plasma, Serum, Urine and Cell culture medi

Protocol
    TotalExoRNA™ Assay Protocol:
  1. Plasma and Serum sample preparation: Prepare plasma samples by 3 centrifugation steps to eliminate red blood cells and cellular debris.
    • 10 min at 300g (save supernatant, discard pellet).
    • 20 min at 1200g (save supernatant, discard pellet).
    • 30 min at 10,000g (save supernatant, discard pellet).
  2. Urine sample preparation:
    • Centrifuge at 16,000g for 20 min at room temperature.
    • Filter by using 0.45 μm filter.
    • Concentrate urine samples by spin concentrator 15-20 times*.
  3. Cell supernatant sample preparation:
    • 10 min at 300g (save supernatant, discard pellet).
    • 20 min at 1600g (save supernatant, discard pellet).
    • 30 min at 10,000g (save supernatant, discard pellet).
    • Concentrate cell supernatant 10-20 fold in spin concentrator*. *The quantity of exosomes could vary between samples. A larger starting amount of sample should be used if the signal is weak.
  4. Reagent preparation:
    • Bead Washing Buffer: Dilute bead washing buffer 5X to 1X with deionized water. Ensure there is no crystal precipitate. NOTE: If crystals are observed, dissolve them by warming up the concentrated 5X Washing Buffer bottle at 37 °C before proceeding with the dilution. Mix 5 mL of 5X Beads Washing Buffer with 20 mL deionized water for a final volume of 25 ml.
    • RNA Washing Buffer Solution: Add into the bottle containing RNA Washing Buffer the volume of pure ethanol (96 % ) indicated on the bottle's label to get the final ethanol concentration of approximately 70 %.
    • Elution Buffer and Lysis Buffer are ready to use.
  5. Exosome binding:
    • Purified Exosomes (Lyophilized Standards): Purified exosomes do not require this binding step. If the samples are purified exosomes, skip to RNA extraction directly.
    • Unfractionated Samples:
      1. Place 0.1 mL up to 1 mL of sample into low-binding tubes (not provided in the kit). Volumes suggested: 0.1 mL up to 0.5 mL for small RNA analysis, 0.5 mL up to 1 mL for mRNA analysis.
      2. Add 1X PBS to the sample to get a final volume of 1 ml. (If you are using 1 mL of plasma, dilution is not necessary).
      3. Add 10 μL of immunobeads.
      4. Incubate sample-immunobead mixture overnight at 4 °C in a rotator.
      5. Centrifuge at room temperature (RT) for 10 min at 5,000g.
      6. Discard the supernatant.
      7. Wash the beads:
        • Add 1 mL of Bead Washing Buffer.
        • Resuspend up and down 10-15 times.
        • Centrifuge at RT for 10 min at 5,000g.
        • Remove the supernatant being careful not to disturb the pellet.
        • Wash beads once again as indicated above.
  6. RNA Extraction:
    • Lysis:
      1. Purified Exosomes: Add 200 μL of Lysis buffer directly to lyophilized exosomes. Resuspend by pipetting and transfer to a fresh tube. Add 500 μL of Lysis buffer to reach a final volume of 700 μL. Incubate for 5 min at room temperature.
      2. Unfractionated Samples: Add 700 μL of Lysis buffer directly on the bead pellet. Dissolve the pellet by pipetting up and down (beads must be totally dissolved). Incubate for 5 min at room temperature.
    • Extraction:
      1. Add 70 μL of 1-Bromo-3-chloropropane (BCP) or 140 μL of pure Chloroform.
      2. Shake 30 sec.
      3. Incubate for 10 min at room temperature.
      4. Incubate 1 min on ice and centrifuge at 12,000g at 4 °C for 10 min. NOTE: Incubation on ice before to centrifuge helps to reduce DNA contamination, which tend to remain in the interphase.
      5. Transfer the top phase (aqueous) to a fresh tube.
      6. Add 2X of ethanol 96 %. Mix by gently inverting 4-5 times. If the top phase volume is 400 μL, add 800 μL of ethanol 96 %.
    • Purification:
      1. Transfer the half volume of the mixture into spin column.
      2. Spin at 14,000g for 30 sec.
      3. Discard the flow-throw.
      4. Add the remaining volume into the same spin column.
      5. Spin at 14,000g for 30 sec. Discard the flow-throw.
      6. Wash column with RNA Washing buffer. Add in the column 400 μL of RNA Washing buffer. Spin at 14,000g for 30 sec. Discard the flow-through. Perform the washing step twice more.
      7. Spin 5 additional min at 14,000g to eliminate ethanol residues from column. Discard the flow-throw.
      8. Remove the tube and transfer the spin column into an elution tube.
      9. Elute the column with 15 μL of Elution buffer. Incubate 5 min at room temperature. Spin 2 min at 200g and 1 min at 14,000g. Keep the flow-through. Eluted RNA is now ready for downstream analysis or for storage at -80 °C
  7. Sensitivity: TotalExoRNATM purified exosome RNA can be quantified and analyzed using the NanoDrop spectrophotometer (Thermo Scientific), although the measured concentration values are likely to end towards the bottom limit of detection of the instrument. For better quantification, we recommend the concomitant use of electropherogram-based technologies (e.g. Bioanalyzer, Agilent Technologies) or fluorimetric technologies (Qubit nano, Thermoscientific). Since most of the RNA contained in extracellular vesicles are small-non-coding RNAs (eg. miRNA), the expected Nanodrop profile, purity and yield are as shown in the representative Figure
  8. TotalExoRNATM allows extraction of high quality of exosome-derived RNAs from low volumes of sample and better performance than competitors. Efficiency of TotalExoRNATM kit was tested vs a competitor kit for RNA extraction from healthy donor plasma derived exosomes.
Reagent Preparation
  • Immunobeads can be stored at +4 °C for up to 8 months.
  • The RNase free columns and elution tubes must be stored at room temperature.
  • All the opened buffers and diluted reagents including the bead washing buffer, RNA washing buffer, lysis buffer and the elution buffer should be stored at +4 °C.
Restrictions For Research Use only
Storage 4 °C
Storage Comment All the reagents are shipped and stored at +4°C for up to 8 months, if unopened. Briefly centrifuge small vials prior to opening. DO NOT FREEZE!
Expiry Date 4-8 months
Supplier Images
Quantification (Q) image for TotalExoRNA™ Overall exosome immunocapture and RNA extraction kit (Biological Fluids/Cell Media, 10 reactions) (ABIN2868142) Expected Nanodrop profile for RNA extracted from immunocaptured exosomes (100 μl of h...
Quantification (Q) image for TotalExoRNA™ Overall exosome immunocapture and RNA extraction kit (Biological Fluids/Cell Media, 10 reactions) (ABIN2868142) Efficiency of TotalExoRNATM kit was tested vs a competitor kit for RNA ext...