RNA Isolation, cDNA Synthesis, and qPCR Analysis

Total RNA was isolated with TRIzol (Invitrogen, Carlsbad, CA, USA) and 2 µg of total RNA was used for cDNA synthesis using a high capacity cDNA reverse transcription kit (Applied Biosystems, Foster City, CA, USA). Purity of isolated total RNA was measured by using the ratio of absorbance at 260 nm and 280 nm (A260/A280), and the ratio of A260/A280 higher than 1.8 was considered to be of acceptable purity. To analyze the gene expression, quantitative PCR was performed using the SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA) and LightCycler96 real-time PCR (Roche, Basel, Switzerland). Ct values were normalized to the human 36B4 gene and relative mRNA expression was calculated versus human 36B4 expression as previously described [36 (link)]. The primer sequences used in the experiment are shown in Table 1.