Superscript 3 direct cdna synthesis system

Total RNA from cells in 24-well cell culture plates was isolated using TRIZOL (Life Technologies, Grand Island, NY, USA) following the manufacturer’s instructions. Total RNA (500 ng) was converted to cDNA by Superscript III Direct cDNA Synthesis System (Life Technologies). Primers were designed to span exon–exon junction in order to preclude the amplification of genomic DNA; primers for human GFAP: forward, 5′-AGGGGGCAAAAGCACCAAAGA-3′, reverse 5′-CTGGGAAAATGA CGCAGTCCAG-3′; for GDNF: forward, 5′-CCCGCCGCAAATATGCCAGA-3′, reverse, 5′-GTTCCTCCTTGGTTT CATAGCCC-3′; for BDNF, forward, 5′-GCCATCCCAAGGTCTAGGTG-3′, reverse, 5′-GTGGGATGGTGGGCATAAGT-3′; for GAPDH; forward 5′-AACAGC GACACCC ACTCC TC-3′, reverse 5′-AGCCAAATTCGTTGTCATACCAGG-3′. The target amplicon sizes were 274, 208, 261, and 100 bp for GDNF, BDNF, GFAP, and GAPDH, respectively. QPCR was done with ViiA Real-time PCR system (ThemoFisher). Data were analyzed with ViiA 7 software with GAPDH as internal control, which using delta delta CT method to calculate gene expression. Gene expression was further normalized to vehicle control, which represented as relative quantity.