TRIzol Reagent

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	- For low 260/230 readings the best approach is to try washing sample (precipitation) with ethanol to desalt it.

Protocol tips
- For low 260/230 readings the best approach is to try washing sample (precipitation) with ethanol to desalt it.

Publication protocol

DuCap and LnCap cells were kindly donated by Matthias Nees (VTT, Finland); Gregor Tevz (APCRC, Australia); and Michelle Hill (UQDI, Australia). DuCap and LnCap cells were cultured in RPMI-1640 growth media (Life Technologies, Australia) supplemented with 10% fetal bovine serum (Life Technologies, Australia) in a humidified incubator (Sanyo MCO-19AIC, Japan) containing 5% CO2 at 37 ºC. Total RNA was extracted by lysing cells with Trizol® reagent (Life Technologies, Australia) before adding chloroform to separate RNA into a clear upper aqueous layer. Then, RNA was precipitated from the aqueous layer and resuspended in 50 µL of RNase-free water. RNA purity and integrity were checked using a NanoDrop Spectrophotometer (Thermo Fisher Scientific, Australia)

For urinary exosomal RNA extraction, microvesicles in the supernatant (after urinary sediment centrifugation) was firstly pelleted by ultracentrifugation (Beckman Coulter OptimaTM XL-100 K, Australia) at 100 000 g, 4ºC for 90 min and resuspended in 200 µL of 10 mM PBS buffer. Then, the Exo-spinTM kit (Cell Guidance Systems, USA) was used accordingly to manufacturer's instructions to isolate exosomes from the pellet. To ensure that extracted RNA was of exosomal origin, the isolated urinary exosomes were treated with RNase A (New England Biolabs, Australia) to degrade all non-exosomal RNA before RNA extraction. Exosomal total RNA was extracted by Trizol® reagent (Life Technologies, Australia) using the procedure as described for RNA extraction from cells.

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