Beacon designer 4

Total cellular RNA was isolated using TRIzol Reagent (cat. 15596026, ThermoFisher Scientific). cDNA was then synthesized from 1 μg of RNA with the RevertAid™ H Minus First Strand cDNA Synthesis Kit (cat. K1632, ThermoFisher Scientific). Gene expression versus β-actin was evaluated by RT-PCR on a MX3000P Real-Time PCR System (Agilent Technology, Milan, Italy). The sequences of the oligonucleotide primers are reported in Table 2. PCR primers were designed using Beacon Designer 4 software (version 4.0, Agilent Technology) from published sequence data stored in the NCBI database. PCR reactions were performed in a total volume of 20 μL, which contained 25 ng of cDNA, 1X Brilliant II SYBR^® Green QPCR Master Mix (cat. 600828, Agilent Technology), ROX Reference Dye (cat. 600804, Agilent Technology), and 600 nM of specific primers. The thermal cycling conditions were 1 cycle at 95 °C for 5 min followed by 45 cycles at 95 °C for 20 s and 60 °C for 30 s. In order to verify the possible coamplification of unspecific targets, melting curves were performed for all of the primer pairs in standard conditions. The data required for carrying out a comparative analysis of gene expression were obtained by means of the 2−(ΔΔCT) method [34 (link)].

Total cellular RNA was isolated using TRIzol Reagent (cat. 15596026, ThermoFisher Scientific). cDNA was then synthesized from 1 µg of RNA with the RevertAid™ H Minus First Strand cDNA Synthesis Kit (cat. K1632, Thermo Fisher Scientific). Gene expression versus β-actin was evaluated by RT-PCR on a MX3000P Real-Time PCR System (Agilent Technology, Milan, Italy). The sequences of the oligonucleotide primers are reported in Table 1. PCR primers were designed using Beacon Designer 4 software (version 4.0, Agilent Technology) from published sequence data stored in the NCBI database. PCR reactions were performed in a total volume of 20 µL, which contained 25 ng of cDNA, 1X Brilliant II SYBR^® Green QPCR Master Mix (cat. 600828, Agilent Technology), ROX Reference Dye (cat. 600804, Agilent Technology), and 600 nM of specific primers. The thermal cycling conditions were 1 cycle at 95 °C for 5 min, followed by 45 cycles at 95 °C for 20 s and 60 °C for 30 s. In order to verify the possible co-amplification of the unspecific targets, the melting curves were performed for all primer pairs in standard conditions. The data required for carrying out a comparative analysis of gene expression were obtained by means of the 2-(∆∆CT) method [33 (link)].