Quantitative PCR (qPCR)

For RT-qPCR, first total RNA was extracted using the RNeasy Plus Mini Kit (Qiagen, Hilden, Germany). RNA was then reverse-transcribed into cDNA using Superscript III transcriptase, according to the manufacturer’s protocol (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA). RT-qPCR was performed using the SYBR Green PCR Master Mix (Applied Biosystems; Thermo Fisher Scientific, Inc., Waltham, MA, USA) on a ROTOR GENE real-time PCR machine (Corbett Robotics, Qiagen, Hilden, Germany). Primers were designed as previously shown [42]; with the sequences freely available from the Entrez Nucleotide database and the Primer3 algorithm for primer design (https://www-genome.wi.mit.edu/cgi-bin/primer/primer3_www.cgi). All primers for qPCR are listed in Additional file 2. PCR volume was 20 μl, and conditions were as follow: initial cycle 50 °C, 2 min, 95 °C, 15 min; 45 cycles at 95 °C, 20 s, 60 °C, 20 s and 72 °C, 20 s; final cycle 72 °C, 30 s. Data were analyzed by the Rotor-Gene software using the comparative ΔΔCt method. The relative copy number was calculated based on the target gene/18S RNA ratio.

All values were expressed as the means ± S.D. Each sample was tested in triplicate.