Quantification of lncRNA fragments by qRT-PCR

For lncRNA detection, the extracted RNA was reverse-transcribed into cDNA with random hexamer primers using the SuperScript^® III First-Strand Synthesis System for RT-PCR (Invitrogen, USA). qRT-PCR was performed using these cDNA products with SYBR^® Green PCR Master Mix (TransGen, China) on an Applied Biosystems 7500 Real-Time PCR system (Applied Biosystems, USA), and β-actin was used as an internal control. The sequences of the gene-specific primers were as follows. β-actin sense: 5′-TTAGTTGCGTTACACCCTT T-3′; antisense: 5′-ACCTTCACCGTTCCAGTTT-3′; LncRNA16: sense: 5′-GATGACAGTCTGCCTCTATCT TAC-3′; antisense: 5′-CTTTGAGCCAAGCAGGTTAT TG-3′. Relative lncRNA levels were calculated by 2^–ΔCT (where ΔCt = Ct(gene) − Ct(β-actin)). The fold change of lncRNA expression in tumor samples versus non-tumor samples was calculated using the 2^–ΔΔCT method.