M mlv reverse transcript reagent

Total RNA was extracted from HCC Huh7 and SNU387 cells with TRIzol (Invitrogen, Carlsbad, CA, USA). cDNA was synthesized using M-MLV Reverse Transcript reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. qPCR was performed using SYBR Green PCR kit (Takara Bio, Inc., Otsu, Japan) and analyzed using an ABI Prism 7500 Real-time PCR system (Applied Biosystems, CA, USA) under the following reaction conditions: 50°C for 2 min; 95°C for 30 sec and followed by 40 cycles of 60°C for 30 sec. All reactions were performed in triplicate. ARK5 mRNA expression was quantified and reported as 2^−ΔΔCq (16 (link)). The expression level of each gene was normalized to the expression level of GAPDH. The primers were as follows: ARK forward, 5′-GAGTCCACTCTATGCATC-3′ and reverse, 5′-GGCCACTATTGAGGACA-3′.