*4.4. Total RNA Isolation for RT-PCR*

SH-SY5Y cells (2.2 <sup>×</sup> 104 cells/mL) were pre-treated in 60 mm cell culture dishes with NE 52-QQ57 (100 nM), then left in serum-free cell culture media for 1 h, followed by stimulation with or without MPP<sup>+</sup> (1 mM) or H2O2 for 18 h, once again in serum-free media. TRIzol (Invitrogen; Burlington, ON, Canada) was employed to extract the total RNA from the cells. 2.5 μg total RNA from each group was reverse-transcribed using a first-strand cDNA synthesis kit (Invitrogen). The following primers were utilised for PCR: GPR4 sense, 5 -CCGTTGTCAAGACCGGGG-3 ; GPR4 anti-sense, 5 -TCCTAGGACCCCCAGAAAGCA-3 ; Bax sense, 5 -CACCAAGGTGCCGGAACTGA-3 ; Bax anti-sense, 5 -AATGCCCATGTCCCCCAATC-3 ; Bcl-2 sense, 5 -ACGACTTCTCCCGCCGCTAC-3 ; Bcl-2 anti-sense, 5 -CCCAGCCTCCGTTATCCTGG-3 ; GAPDH sense, 5 -GCAGTGGCAAAGTGGAGATTG-3 ; and GAPDH anti-sense, 5 - TGCAGGATGCATTGCTGACA-3 . Then, adopting the previously mentioned primers, the cDNA was amplified through PCR [45]. GAPDH was employed as an internal control to evaluate the relative levels of expression of other genes. PCR products were analysed on 1.0–1.2% agarose gels stained with GelRed (Sigma-Aldrich; St. Louis, MO, USA). The gels were photographed and, utilising ImageJ (NIH) software, the pixel intensity for each band in the photographs was measured and normalised to the band intensity of the GAPDH mRNA, to quantify its relative expression.
