Quantifying miR-3650 Expression in Hepatocellular Carcinoma

Total RNA was isolated from fresh-frozen tissues and HCC cells using TRIzol reagent (Invitrogen, NY, USA) as per the manufacturer’s instructions. The quantity of RNA samples was measured by a NanoDropTM 1000 spectrophotometer (ThermoFisher Scientific, Waltham, MA, USA). 1 μg total RNA and All-in-one^TM First-Strand cDNA Synthesis Kit (GeneCopoeia, Rockville, USA) were used to generate cDNA, which was subjected to RT-PCR using All-in-One™ miRNA qRT-PCR Detection Kit (Gene-Copoeia, Carlsbad, CA, USA). PCR was performed on a Roche Light Cycler 480 instrument (Roche, Basel, Switzerland) according to a standard method as described previously [30 (link)]. The relative expression of miR-3650 was calculated using the 2^−ΔΔCt method and U6 was used as the endogenous control. The primer sequences used in this study are shown in supplementary Table S1.