RNA Isolation Methods - Diverse Biofluids
To evaluate performance of different extra cellular RNA isolation methods in biofluids Overall design: Serum and plasma were collected from healthy adult donors, 10 female and 10 male. Three Core exRNA isolation methods (ExoRNeasy (Qiagen), miRNeasy (Qiagen), and miRCURY Biofluids (Exiqon)) were tested in all labs, and 7 methods (Exosomal RNA Purification Kit (Norgen Biotek) w/o Amicon 3K, Exosomal RNA Purification Kit (Norgen Biotek) w/ Amicon 3K, Ultra + miRNeasy, Millipore + miRNeasy, ME Kit (New England Peptide) + miRNeasy, ExoQuick+Seramir (SBI), miRCURY Exosome Isolation + miRCURY Biofluids (Exiqon)) were tested in a subset of labs. CCCM was collected from three cell types in three different labs: human embryonic stem cells (hESCs); primary rat cardiomyocytes (NRVM); and cholangiocarcinoma cells (KMBC). exRNA isolation experiments in triplicate for the supernatants from each cell type. Bile was collected from three patients with cholangiocarcinoma using four methods: ExoRNeasy; Ultra; Millipore; and ExoQuick. exRNA isolation experiments in triplicate for the bile from each donor using five methods: ExoRNeasy; miRNeasy; Exiqon; Ultra; and Millipore. Urine was collected from healthy adult donors, 10 female and 10 male (the same donors as for plasma and serum).exRNA isolation experiments in triplicate for the urine from each donor using five methods: ExoQuick; ExoRNeasy; Homebrew; Millipore; and Ultra . Small RNA sequencing libraries were constructed using the NEB Next small RNA library kit.