RNA isolation and qRT-PCR.
Total RNA was isolated using TRIZOL reagent (Life Technologies, USA) according to the method of Chomczynski and Sacchi (1987). The integrity of the samples was verified by 1% agarose gel electrophoresis and the RNA amount in each sample was measured by spectrophotometry, as described (Caputi, et al., 2021a).
RNA samples were subsequently subjected to DNase treatment and converted to cDNA using the GeneAmp RNA PCR kit (Life Technologies, Italy) according to the manufacturer’s protocol.
The qRT-PCR analysis was performed on a StepOne Real-Time PCR System (Life Technologies, Monza, Italy) using SYBR Green PCR MasterMix (Life Technologies, Italy), as previously reported (Caputi, et al., 2021b). Relative expression of the different gene transcripts was calculated by the Delta-Delta Ct (DDCt) method and converted to relative expression ratio (2-DDCt) for statistical analysis (Livak & Schmittgen, 2001). All data were normalised to the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The primers, designed using Primer 3 (Rozen & Skaletsky, 2000) were used for PCR amplification.
pDYN forward 5′− CCTGTCCTTGTGTTCCCTGT-3′; pDYN reverse 5′- AGAGGCAGTCAGGGTGAGAA-3′; KOR forward 5′- TTGGCTACTGGCATCATCTG-3′; KOR reverse 5′- ACACTCTTCAAGCGCAGGAT-3′; CRF forward 5′-GCAGCGGGACTTCTGTTGA-3′; CRF reverse 5′-CGCAGCCGTTGAATTTCTTG-3′; CRFR1 forward 5′- TGCCAGGAGATTCTCAACGAA-3′; CRFR1 reverse 5′- AAAGCCGAGATGAGGTTCCAG-3′; GAPDH forward 5′- AGACAGCCGCATCTTCTTGT-3′; GAPDH reverse 5′- CTTGCCGTGGGTAGAGTCAT-3′.