This protocol describes how to isolate exosomal RNA from urine by using the Qiagen ExoRNeasy maxi kit.
Method Article
Isolation of exosomal RNA from urine using the Qiagen ExoRNeasy maxi kit
https://doi.org/10.1038/protex.2017.081
This work is licensed under a CC BY-NC 3.0 License
This protocol has been posted on Protocol Exchange, an open repository of community-contributed protocols sponsored by Nature Portfolio. These protocols are posted directly on the Protocol Exchange by authors and are made freely available to the scientific community for use and comment.
posted
You are reading this latest protocol version
This protocol describes how to isolate exosomal RNA from urine by using the Qiagen ExoRNeasy maxi kit.
exRNA
extracellular vesicles
exosomes
urine
RNA isolation
ExoRNeasy
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.
ExoRNeasy Maxi kit (Qiagen, 77064)
RNeasy MinElute Columns (Qiagen, part of miRNeasy micro kit - 217084)
Urine (pre-cleared by centrifugation at 2000 x g for 10 min)
Chloroform (Sigma-Aldrich, 319988)
100% ethanol (Koptec, V1016)
RNase-free water (Ambion, AM9937)
Phase lock gel tubes, 2 mL (VWR, 10847-802)
Microfuge
Centrifuge
Microfuge tubes, 1.5 mL
1 PLG makes separation of the aqueous phase from the interface easier, and thus is particularly useful for large numbers of samples or less experienced personnel, but it is expensive.
2 A spike-in control can be added here. For example, add 3 μL of the miRNeasy Serum/Plasma Spike-In Control (the manufacturer’s instructions produce a working solution of 1.6 x 108 copies/μL, so if you add 3 μL of the Spike-In Control and elute your exRNA in a final volume of 30 μL, the theoretical concentration of the Spike-In Control in the final exRNA sample would be 1.6 x 107 copies/μL).
3 The centrifuge must be above 20°C so that excessive precipitation does not occur.
4 After centrifugation, carefully remove the RNeasy MinElute spin column from the collection tube so that the column does not contact the flow-through. Otherwise, carryover of ethanol will occur.
5 To avoid damage to their lids, place the spin columns into the centrifuge with at least one empty position between columns. Orient the lids so that they point in a direction opposite to the rotation of the rotor (e.g., if the rotor rotates clockwise, orient the lids counterclockwise). It is important to dry the spin column membrane, since residual ethanol may interfere with downstream reactions. Centrifugation with the lids open ensures that no ethanol is carried over during RNA elution.
6 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.
This protocol was modified from the manufacturer's instructions for the ExoRNeasy Maxi kit.
This work was supported by grants U01 HL126494 and UH3 TR000906 from the National Institutes of Health.
The authors declare no competing financial interests.
This protocol has been posted on Protocol Exchange, an open repository of community-contributed protocols sponsored by Nature Portfolio. These protocols are posted directly on the Protocol Exchange by authors and are made freely available to the scientific community for use and comment.
posted
You are reading this latest protocol version