Mj mini personal thermal cycler apparatus

Total RNA was extracted with TRIzol reagent (Sigma-Aldrich) according to the manufacturer's protocol and 2 µg were reverse-transcribed with the Omniscript RT kit (Qiagen, Italy) using random primers (1 mM) at 37°C for 1 h. Real time PCR was performed in triplicate in 20 mL reaction volumes using the Power SYBER Green PCR Master Mix (Applied Biosystems, USA). All primers were purchased from Invitrogen. Real time PCR reactions were carried out in a MJ Mini™ Personal Thermal Cycler apparatus (Bio-Rad Laboratories, USA). Melting curves were obtained by increasing the temperature from 60 to 95°C with a temperature transition rate of 0.5°C/s. The comparative threshold cycle number (C_T) method was used to assess the relative quantification of gene expression. The fold change of the target gene was calculated as 2^-ΔΔCT. The internal controls were GAPDH for IL-1, TNF-α, Cox2, and U6 for miR-142-3p.

Total RNA was extracted with TRIzol reagent according to the manufacturer’s protocol (Sigma). Two micrograms of the isolated RNA were reverse-transcribed with the Applyed Biosystem (Thermo fisher) kit. Real-time PCR was carried out in triplicate in 20 μl reaction volumes using the Power SYBER Green PCR Master Mix (Applied Biosystems). PCR primers for human c-fos mRNA are the following: forward 5′-CGGGCTTCAACGCAGACTA-3′; reverse 5′-GGTCCGTGCAGAAGTCCTG-3′. Real-time PCR reactions were carried out in a MJ Mini Personal Thermal Cycler apparatus (Bio-Rad Laboratories). Melting curves were obtained by increasing the temperature from 60 to 95 °C with a temperature transition rate of 0.5 °C s⁻¹. Melting curves of final PCR products were analyzed (OpticonMonitor 3 Bio-Rad).