MALAT1 expression analysis by reverse transcription-quantitative PCR (RT-qPCR)

Total RNA was prepared from liver tumor tissues and cancer cells using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.). A PrimeScript™ RT Reagent kit with genomic DNA Eraser (Takara Bio, Inc.) was used to reverse transcribe RNA into cDNA at 42°C for 1 h according to the manufacturer's instructions. Subsequently, qPCR was performed using an ABI Prism 7900 kit (Applied Biosystems; Thermo Fisher Scientific, Inc.). The following thermocycling conditions were used for qPCR: Initial denaturation at 95°C for 30 sec; followed by 40 cycles at 95°C for 5 sec and at 60°C for 30 sec. The following primers were used for qPCR: MALAT1 forward, 5′-ATACCTAACCAGGCATAACA-3′ and reverse, 5′-AGTAGACCAACTAAGCGAAT-3′; and GAPDH forward, 5′-GACCTGACCTGCCGTCTAG-3′ and reverse, 5′-AGGAGTGGGTGTCGCTGT-3′. The 2^−ΔΔCq method was used to determine relative MALAT1 expression levels (15), which were normalized to the internal reference gene GAPDH.