RNA was extracted using the RNAqueous™-Micro Total RNA Isolation Kit (ThermoFisher Scientific), followed by a DNAse treatment made with the TURBO DNA-free™ Kit (ThermoFisher Scientific) and finally purified a second time using the RNeasy MinElute Cleanup Kit (Qiagen). All steps were done according to the manufacturer's protocol.
Real-time PCR was performed using the SYBR Green Supermix (Biorad) with a Biorad’s thermal cycler using the following profile: 95 °C for 10 min; 40 cycles of amplification with successively 95 °C for 15 s, 60 °C for 10 s, and 72 °C for 20 s; one cycle for melting curve analysis with an acquisition every 0.5 °C from 65 °C to 95 °C to verify the presence of a single product. Each assay included a no-template control for each primer pair, and five successive dilutions to determine the Ct values and the reaction efficiencies. Real-time PCR reactions were done in triplicate. Gene expression level was normalized using Ci-actin as reference gene as previously described31 (link)–33 (link). Primer pair used for Ci-caspase 8/10 are: forward 5’-AAGACTGCTTTGTGTGCGTG-3’, reverse 5’-GGCAGGCTTGGAAGAAAAATAT-3’. PCR products length were between 140 and 160 bp.
Real-time PCR was performed using the SYBR Green Supermix (Biorad) with a Biorad’s thermal cycler using the following profile: 95 °C for 10 min; 40 cycles of amplification with successively 95 °C for 15 s, 60 °C for 10 s, and 72 °C for 20 s; one cycle for melting curve analysis with an acquisition every 0.5 °C from 65 °C to 95 °C to verify the presence of a single product. Each assay included a no-template control for each primer pair, and five successive dilutions to determine the Ct values and the reaction efficiencies. Real-time PCR reactions were done in triplicate. Gene expression level was normalized using Ci-actin as reference gene as previously described31 (link)–33 (link). Primer pair used for Ci-caspase 8/10 are: forward 5’-AAGACTGCTTTGTGTGCGTG-3’, reverse 5’-GGCAGGCTTGGAAGAAAAATAT-3’. PCR products length were between 140 and 160 bp.
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