Quantifying SERPINB3 and HIF-2α Expression in Liver Tumors

RNA extraction, complementary DNA synthesis, quantitative real-time PCR (Q-PCR) reactions were performed as previously described [30 (link)]. SERPINB3 and HIF-2α mRNA levels were measured by Q-PCR, using the SYBR® green method as described [30 (link)]. The amplification mix was prepared using Roche LightCycler FastStart DNA MasterPLUS SYBR Green I kit following manufacturer's instructions and real-time PCR was performed using LightCycler instrument. Oligonucleotide sequence of primers used for RT-PCR were: sense, 5′-GCAAATGCTCCAGAAGAAAG-3′, reverse 5′-CGAGGCAAAATGAAAAGATG-3′ (for human SERPINB3); sense, 5′-GGAGCCACGGTCTCTCAGTA-3′, reverse 5′-TGCATCTATGGGGATGAGAA-3′ (for human SERPINB4); sense, 5′-CGCTAGACTCCGAGAACAT-3′, reverse 5′-GGCTTGAACAGGGATTCAGT-3′ (for human HIF2α). Gliceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as internal reference and co-amplified with target samples using identical Q-PCR conditions. In particular, expression of SERPINB3 and HIF-2α mRNA was also assessed by Q-PCR in 67 frozen liver tumor samples displaying different patterns of SERPINB3 expression (SERPINB3 negative/low expression or SERPINB3 high expression) [30 (link)]. Samples were run in triplicate and mRNA expression was generated for each sample. Specificity of the amplified PCR products was determined by melting curve analysis and confirmed by agarose gel electrophoresis.