One 10mL archived urine sample per participant from sampling day 1 was thawed on ice and then stabilized with 1mL EDTA. Samples were inverted five times to mix well and centrifuged (3,000 × G for 30 min at 4°C). Urinary supernatant was removed and set aside while the remaining pellet was washed with 1mL PBS and centrifuged (12,000 × G for 10 minutes at 4°C). After the spin, the 1mL PBS supernatant was discarded and Ambion mirVana PARIS miRNA (Ambion, Austin, TX) kit-sourced denaturing buffer was mixed thoroughly with the retained pellet. 1mL of the urinary supernatant was added back to the denatured pellet and RNA isolation proceeded according to the Ambion mirVana PARIS miRNA kit manufacturer’s protocol. Potential DNA contamination was removed using Ambion’s DNA-free DNA Removal kit (Ambion, Austin, TX) per the manufacturer’s protocol. Finally, RNA samples were cleaned and concentrated using Qiagen’s RNeasy MinElute Cleanup Kit (Qiagen, Ltd) following the manufacturer’s protocol.
Total RNA concentrations were determined on a NanoDrop spectrophotometer [NanoDrop Technologies, Wilmington, DE]. Urinary miRNA samples were profiled using TaqMan Array Human miRNA Panel A (4398977) [LifeTechnologies Carlsbad, CA] covering 384 human miRNAs following manufacturer instructions. The top three most frequently expressed miRNAs were validated by RT-PCR.
Total RNA concentrations were determined on a NanoDrop spectrophotometer [NanoDrop Technologies, Wilmington, DE]. Urinary miRNA samples were profiled using TaqMan Array Human miRNA Panel A (4398977) [LifeTechnologies Carlsbad, CA] covering 384 human miRNAs following manufacturer instructions. The top three most frequently expressed miRNAs were validated by RT-PCR.