Gotaq probe systems

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.

Total RNA was extracted from cells by using TRI Reagent (Sigma). The amount and purity of RNA were determined spectrophotometrically. cDNA synthesis was obtained by incubating 2 µg of total RNA with 4 U/µL of M-MLV reverse transcriptase (Promega, San Luis Obispo, California) according to the manufacturer’s instructions.
Quantitative real time PCR (qPCR) was performed 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 of 95° for 15 s and 60 °C for 60 s. mRNA was quantified with the ΔΔCt method as described²³ (link). mRNA levels were normalised to β-2 microglobulin and β-actin as endogenous controls. Primer sequences are reported in Table 1.

Total RNA was isolated from cells by using TRI Reagent (Sigma). 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 [27 (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 ∆∆Ct method as described [33 (link)]. mRNA levels were normalized to 18S as an endogenous control. The primer sequences used are listed in Table 1.