This is a protocol preprint. Preprints are preliminary reports that have not undergone peer review. They should not be considered conclusive, used to inform clinical practice, or referenced by the media as validated information.
Method Article
Isolation of exosomal RNA from cell culture supernatant using the System Biosciences Exoquick Seramir tissue culture kit
Saumya Das
Cardiovascular Research Center, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA
Corresponding Author
Louise C. Laurent
Department of Reproductive Medicine, University of California, San Diego, La Jolla, CA, USA
Corresponding Author
Tushar Patel
Mayo Clinic Florida, Jacksonville, FL, USA
Corresponding Author
License:
This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License
This protocol describes how to isolate exosomal RNA from cell culture supernatant using the Exoquick Seramir tissue culture kit.
Keywords
exRNA, extracellular vesicles, exosomes, RNA isolation, Exoquick, SeraMir
Extracellular RNAs (exRNAs) have been found to play an important role in intercellular communication in the body. exRNAs are present in biofluids, in complexes with lipoproteins and ribonucleoproteins, and in extracellular vesicles such as exosomes and microvesicles. Further study and characterization of exRNAs and their carriers could lead to identification of new biomarkers and have potential for development of novel therapeutics.
Exoquick Seramir tissue culture kit (Systems Biosciences, RA800-TC1)
100% ethanol (VWR, 89125-186)
Microfuge
Microfuge tubes, 1.5 mL
- Harvest cell culture medium from 80% confluent cells grown in serum-free medium.
- Optional: Spin media at 500-1,000 x g for 10 min.
- Pre-clear medium by centrifuging at 2000 x g for 10 min, then filter using a 0.22 µm PES Steriflip membrane and freeze at -80°C until needed.
- Thaw 4 mL of cell culture supernatant on ice.
- Add 0.8 mL of Exoquick-TC and mix well by inversion 3 times.
- Incubate at 4°C overnight.
- Centrifuge at 16,000 x g for 2 min, remove supernatant and keep the exosome pellet.
- Add 350 µL Lysis buffer to pellet, vortex for 15 s, and incubate at room temperature for 5 min.
- Add 200 µL of 100% ethanol and vortex for 10 s.
- Transfer all (600 µL) to spin column and centrifuge at 16,000 x g for 1 min (or until the solution has flowed through).
- Discard flow through.
- Add 400 µL of wash buffer and centrifuge at 16,000 x g for 1 min, then discard flow-through.
- Repeat step 12 once more.
- Discard flow-through and centrifuge at 16,000 x g for 2 min to dry column.
- Transfer spin column to new collection tube and add 14 µL or 30 µL RNase-free water or Elution buffer directly to the membrane.1
- Centrifuge at 400 x g for 2 min.2
- Centrifuge at 16,000 x g for 1 min.
1 This volume was selected to match that of other kits to enable fair comparisons. As little as 10 μL RNase-free water can be used for elution if a higher RNA concentration is required, but the yield will be reduced by approximately 20%. Do not elute with less than 10 μL RNase-free water, as the spin column membrane will not be sufficiently hydrated. The dead volume of the RNeasy MinElute spin column is 2 μL: elution with 14 μL RNase-free water results in a 12 μL eluate.
2 Centrifuging at a low speed first helps the solvent wet the surface of the membrane prior to the full speed centrifuging step. This results in a better yield / RNA recovery from the membrane.
This protocol was modified from the manufacturer's instructions for the Exoquick Seramir kit.
KD, BS and SD were supported by NIH grant UH3TR000901. SS and LL were supported by U01 HL126494 and UH3 TR000906. IY and TP were supported by UH3 TR000884.
Version History
CURRENT STATUS: Posted
Version 1
Posted 16 Jun, 2017
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Method Article
Isolation of exosomal RNA from cell culture supernatant using the System Biosciences Exoquick Seramir tissue culture kit
Saumya Das
Cardiovascular Research Center, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA
Corresponding Author
Louise C. Laurent
Department of Reproductive Medicine, University of California, San Diego, La Jolla, CA, USA
Corresponding Author
Tushar Patel
Mayo Clinic Florida, Jacksonville, FL, USA
Corresponding Author
Version History
CURRENT STATUS: Posted
Version 1
Posted 16 Jun, 2017
Metrics
Comments: 0
HTML Views: ...
License:
This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License
This protocol describes how to isolate exosomal RNA from cell culture supernatant using the Exoquick Seramir tissue culture kit.
Extracellular RNAs (exRNAs) have been found to play an important role in intercellular communication in the body. exRNAs are present in biofluids, in complexes with lipoproteins and ribonucleoproteins, and in extracellular vesicles such as exosomes and microvesicles. Further study and characterization of exRNAs and their carriers could lead to identification of new biomarkers and have potential for development of novel therapeutics.
Exoquick Seramir tissue culture kit (Systems Biosciences, RA800-TC1)
100% ethanol (VWR, 89125-186)
Microfuge
Microfuge tubes, 1.5 mL
- Harvest cell culture medium from 80% confluent cells grown in serum-free medium.
- Optional: Spin media at 500-1,000 x g for 10 min.
- Pre-clear medium by centrifuging at 2000 x g for 10 min, then filter using a 0.22 µm PES Steriflip membrane and freeze at -80°C until needed.
- Thaw 4 mL of cell culture supernatant on ice.
- Add 0.8 mL of Exoquick-TC and mix well by inversion 3 times.
- Incubate at 4°C overnight.
- Centrifuge at 16,000 x g for 2 min, remove supernatant and keep the exosome pellet.
- Add 350 µL Lysis buffer to pellet, vortex for 15 s, and incubate at room temperature for 5 min.
- Add 200 µL of 100% ethanol and vortex for 10 s.
- Transfer all (600 µL) to spin column and centrifuge at 16,000 x g for 1 min (or until the solution has flowed through).
- Discard flow through.
- Add 400 µL of wash buffer and centrifuge at 16,000 x g for 1 min, then discard flow-through.
- Repeat step 12 once more.
- Discard flow-through and centrifuge at 16,000 x g for 2 min to dry column.
- Transfer spin column to new collection tube and add 14 µL or 30 µL RNase-free water or Elution buffer directly to the membrane.1
- Centrifuge at 400 x g for 2 min.2
- Centrifuge at 16,000 x g for 1 min.
1 This volume was selected to match that of other kits to enable fair comparisons. As little as 10 μL RNase-free water can be used for elution if a higher RNA concentration is required, but the yield will be reduced by approximately 20%. Do not elute with less than 10 μL RNase-free water, as the spin column membrane will not be sufficiently hydrated. The dead volume of the RNeasy MinElute spin column is 2 μL: elution with 14 μL RNase-free water results in a 12 μL eluate.
2 Centrifuging at a low speed first helps the solvent wet the surface of the membrane prior to the full speed centrifuging step. This results in a better yield / RNA recovery from the membrane.
This protocol was modified from the manufacturer's instructions for the Exoquick Seramir kit.
KD, BS and SD were supported by NIH grant UH3TR000901. SS and LL were supported by U01 HL126494 and UH3 TR000906. IY and TP were supported by UH3 TR000884.