Evagreen mix

RNA was isolated by TRIzol–chloroform extraction. RNA was reverse-transcribed using a GoScript reverse transcription kit (Promega). qPCR was performed with a final amount of 20 ng of cDNA and EvaGreen Mix (Bio-Rad) with primer pairs for either mouse Gapdh (forward, GCTCATGACCACAGTCCAT; reverse, GTCATCATACTTGGCAGGTTT), mouse Arl6ip1 (forward, GCTCTAATAAATGGACCACTG; reverse, GCACAAATGTCACAATCAGGT), human GAPDH (forward, GAAGGCTGGGGCTCATTT; reverse, GGACTGTGGTCATGAGTC) or human ARL6IP1 (forward, GCTCCAATAAATGGACCACTGA; reverse, GGAAGTCACTATCAGGTAGGT) on a CFX96 Touch Real-Time PCR detection system (Bio-Rad).

Total RNA was extracted using the AURUM total RNA Mini Kit (BioRad), according to the manufacturer’s instructions, and reverse transcribed into cDNA using iScript cDNA Synthesis Kit (BioRad). cDNA was amplified using EvaGreen mix (BioRad) on a CF96 Touch real-time PCR (BioRad). Primers sequences were as follows:
CXCR1: forward 5′-TGCATCAGTGTGGACCGTTA-3′ and reverse: 5′-TGTCATTTCCCAGGACCTCA-3′; CXCR2: forward 5′-TGCATCAGTGTGGACCGTTA-3′ and reverse 5′-CCGCCAGTTTGCTGTATTG-3′ (Maxwell et al., 2007 (link)); GFAP: forward 5′-ATCAACTCACCGCCAACA-3′ and reverse 5′-CGACTCAATCTTCCTCTCCAG-3′; GROα (CXCL1): forward 5′-CTGGCTTAGAACAAAGGGGCT-3′ and reverse 5′-TAAAGGTAGCCCTTGTTTCCCC-3′; GROβ (CXCL2): forward 5′-ACAGTGTGTGGTCAACATTTCTC-3′ and reverse 5′-TCTGCTCTAACACAGAGGGAA-3′; GROγ (CXCL3): forward 5′- CCGAAGTCATAGCCACACTCA-3′ and reverse 5′-CTCTGGTAAGGGCAGGGACC-3′; IL-8 (CXCL8): forward 5′-CTTGGCAGCCTTCCTGATTT-3′ and reverse 5′-AACCCTCTGCACCCAGTTTT-3. Levels of target genes in each sample were normalized on the basis of GAPDH and 28S amplification and reported as relative values (Gritti et al., 2014 (link)).