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′.