Circulating and exosomal RNA were purified from plasma using Plasma/Serum Circulating and Exosomal RNA Purification kit (Norgen Biotek Corp., Canada), according to manufacturer instructions. In order to control extraction efficiency, a panel contained 5 spiked in probes (ath-miR-159a, cel-miR-248, cel-miR-254, osa-miR-414, osa-miR-442; IDT Technologies, USA) was added after second lysis buffer incubation. After purification, RNA portion was concentrated using RNA Clean-Up and Concentration kit (Norgen Biotek Corp., Canada), following manufacturer instructions.
Label
not provided
Label protocol
Extraction product (3 uL) was analyzed using nCounter Human v3 miRNA Expression Assay (NanoString Technologies), according to the manufacturer’s instructions.
Hybridization protocol
according to the manufacturer’s instructions.
Scan protocol
according to the manufacturer’s instructions.
Data processing
Raw data was analysed with NanoString nSolver 4.0, using default quality control standards. For normalization, spike in oligos (cel-miR-248 and cel-miR-254) were used. Following manufacturer’s instructions, only targets that presented average raw count above 100 were considered for further differential expression analysis in Partek Genomics Suite 7.0. Principal component analysis plot was also generated with the same software with batch effects removed.
