Profiling Small RNA Expression in Osteosarcoma

Total RNA was extracted from samples from 10 OS patients and 10 healthy controls using phenol-chloroform (TRIzol; Invitrogen; ThermoFisher Scientific, Inc., Waltham, MA, United States). The quality of RNA was assessed by capillary electrophoresis (Agilent Technologies, Inc., Santa Clara, CA, United States). Libraries for small RNA sequencing were prepared using NEB kits (New England Biolabs, Inc., Ipswich, MA, United States). qRT-PCR with SYBR-Green (Takara, Osaka, Japan) to detect CDC5L, CUL1, CXCL10, EIF2AK2, POLR2B, PTEN, STAT1, and TBP expression levels, GAPDH was applied as a house keeping gene. The reaction was performed via 40 amplification cycles using the following protocol: 95°C for 3 min, 95°C for 45 s, 55°C for 15 s, and 72°C for 50 s. Primers used in PCR were shown in Supplementary Table S1. Samples were analyzed in triplicate, and gene expression was quantified by normalizing target gene expression to that of the internal control using the 2^−ΔΔCt formula.