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 urine using ultracentrifugation and the Qiagen miRNeasy Micro kit
Louise C. Laurent
Department of Reproductive Medicine, University of California, San Diego, La Jolla, CA, 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 urine by using ultracentrifugation for exosomal enrichment followed by the Qiagen miRNeasy Micro kit for RNA isolation.
Keywords
exRNA, extracellular vesicles, exosomes, urine, ultracentrifugation, RNA isolation, miRNeasy 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.
Urine (pre-cleared by centrifugation at 2000 x g for 10 min)
Phosphate-buffered saline (PBS)
Ultracentrifuge (Beckman Coulter, Optima MAX – XP Ultracentrifuge)
Swinging bucket rotor (MLS-50)
Ultracentrifuge tubes (Beckman Coulter, 326819)
- Thaw samples on ice.
- Transfer 0.5 mL of urine into an ultracentrifuge tube.
- Bring up volume to fill the ultracentrifuge tube with PBS (3 mL).
- Centrifuge at 100,000 x g for 70 min.
- Discard 3 mL of supernatant.
- Add 3 mL of fresh PBS.
- Centrifuge at 100,000 x g for 70 min.
- Remove and discard 3.3 mL of supernatant.
- Resuspend the leftover solution (usually about 200 µL).
- Freeze at -80°C until needed.
- Isolate RNA using the miRNeasy Micro kit.
This work was supported by grants U01 HL126494 and UH3 TR000906 from the National Institutes of Health.
Version History
CURRENT STATUS: Posted
Version 1
Posted 16 Jun, 2017
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Method Article
Isolation of exosomal RNA from urine using ultracentrifugation and the Qiagen miRNeasy Micro kit
Louise C. Laurent
Department of Reproductive Medicine, University of California, San Diego, La Jolla, CA, 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 urine by using ultracentrifugation for exosomal enrichment followed by the Qiagen miRNeasy Micro kit for RNA isolation.
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.
Urine (pre-cleared by centrifugation at 2000 x g for 10 min)
Phosphate-buffered saline (PBS)
Ultracentrifuge (Beckman Coulter, Optima MAX – XP Ultracentrifuge)
Swinging bucket rotor (MLS-50)
Ultracentrifuge tubes (Beckman Coulter, 326819)
- Thaw samples on ice.
- Transfer 0.5 mL of urine into an ultracentrifuge tube.
- Bring up volume to fill the ultracentrifuge tube with PBS (3 mL).
- Centrifuge at 100,000 x g for 70 min.
- Discard 3 mL of supernatant.
- Add 3 mL of fresh PBS.
- Centrifuge at 100,000 x g for 70 min.
- Remove and discard 3.3 mL of supernatant.
- Resuspend the leftover solution (usually about 200 µL).
- Freeze at -80°C until needed.
- Isolate RNA using the miRNeasy Micro kit.
This work was supported by grants U01 HL126494 and UH3 TR000906 from the National Institutes of Health.