Quantitative Real-Time PCR for Gene Expression

Total RNA was isolated from cells by using TRI Reagent (Sigma, Milan, Italy). The amount and purity of RNA were determined spectrophotometrically. cDNAwas obtained by incubating 2 μg of total RNA with 4 U/μL of M-MLV reverse transcriptase (Promega, San Luis Obispo, CA, USA) according to the manufacturer’s instructions. Quantitative real time PCR (qPCR) was performed as reported in [51 (link)] using the GoTaq^® Probe Systems (Promega). The qPCR analysis was carried out in triplicate using an Applied Biosystems 7500 Sequence Detector with the default PCR setting: 40 cycles at 95 °C for 15 s, 60 °C for 60 s. mRNA was quantified with the DDCt method as described [52 (link)]. mRNA levels were normalized to β2 microglobulin as an endogenous control. The primer sequences used are listed in Table 1.