Quantifying mRNA Levels in HCC Cells

Total RNA was isolated from HCC cells and patient tissues using TRIzol reagent (Invitrogen). cDNA was synthesized from total RNA using the ImProm-II™ Reverse Transcriptase system (Promega) according to the manufacturer’s protocol and a previous study.^{56 (link),57 (link)} The mRNA levels were measured by quantitative real-time PCR (RT-qPCR), which was performed in an ABI 7500 Real-time PCR system using SYBR Green PCR Master Mix (Applied Biosystems). The qPCR primers used are as follows: 5′-CCCGAAGAAGACGTAGAAGATGAC-3′ and 5′-CCCGAAGAAGAGGTGGAGGACGAC-3′ (Tbx3), 5′-CCACCAAAGTCACGCTGAA-3′ and 5′–TGCTTGGATTCCAGAAACG-3′ (E-cadherin), 5′-CGGAACAAGGAGAAGAG CAA-3′ and 5′- GCTCAGCCACCTTCTGTTTTA-3′ (HDAC5), and 5′-TCCATGACAAC TTTGGTATCG-3′ and 5′-TGTAGCCAAATTCGTTGTCA-3′ (GAPDH). Serial 1 to 10 dilutions of plasmid DNA (pIRES-Tbx3, pcDNA-E-cadherin, pCRII-GAPDH) were used as standards for the absolute quantification of each cDNA. The amount of target gene expression could be calculated from the standard curve, which plotted the cycle threshold (Ct) value and the corresponding value of the amount of input DNA.^{58 (link)}