Isolating RNA From Exosomes

April 30, 2018

Newcastle University
Academic Haematology
Research Associate

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Company:

ThermoFisher

Product Name:

Total Exosome RNA & Protein Isolation Kit

Catalog Number:

4478545

The Total Exosome RNA & Protein Isolation Kit can be used to isolate Total RNA and protein from exosomes. In our lab, we use the Total Exosome RNA & Protein Isolation Kit to isolate RNA from exosomes derived from both cell-conditioned media and also serum.

Experimental Design and Results Summary

Application

RNA isolation from serum and cell-conditioned media exosomes

Starting Material

Exosomes isolated from serum and cell-conditioned media

Protocol Overview

After fully re-suspending the exosome pellet, particle-free PBS is added to make the Volume up to 200ul. One volume of denaturing solution is then added and the sample incubated for 5 minutes on ice. One volume of phenol:chloroform is then added and the sample thoroughly mixed by vortexing for 1 minute. The sample is then centrifuged at maximum speed for 5 minutes to separate aqueous and organic phases, and the aqueous phase is then carefully removed to a new tube. to this, 1.25 volumes of 100% ethanol are added, and the lysate/ethanol mixture is passed through a separation column supplied with the kit by centrifugation for 15s at maximum speed. The flow-through is then discarded and the column is washed by a series of wash buffers. Finally, RNA is eluted by two sequential additions of 50ul elution buffer, which has been pre-heated to 95C, followed by centrifugation for 30s.

Tips

Pre-heat some of the buffers to 37C to remove precipitates which can form during storage

Results Summary

Successfully isolates total RNA which can be quantified using the bioanalyzer.

DOI or PMID #

N/A

Additional Notes

N/A

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Summary

The Good

Effective product that is quick to use and doesn't require specialist equipment.

The Bad

Requires harmful chemicals and the eluate volume is high (100ul), meaning RNA can sometimes require concentration for downstream applications

The Bottom Line

Effective product for simultaneous isolation of total RNA and protein from exosomes.

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