RNA extraction and real-time polymerase chain reaction
Total RNA was extracted with TRI-reagent from control or U50,488H
treated κ-Neuro-2A cells according to manufacturer’s instructions. Total
RNA (1 μg) was used as template for cDNA synthesis using SuperScript II
reverse transcriptase (Thermo Fisher). The following primers were
designed for Real Time-PCR: Becn1,(forward):5’-GGCCAATAAGATGGGTCTGA-3’; (reverse)
5’-GCTGCACACAGTCCAGAAAA-3’; for ATG5, 5’ (forward)
AAGTCTGTCC-TTCCGCAGTC-3’; (reverse) GAAGAAAGTTATCTGGGTAGCTCA-3’; forGAPDH ( forward) 5’-TGTGTCCGTCGTGGATCTGA-3’, (reverse)
5’-CCTGCTTCACCACCTTCT-TGA-3’, using a ΜΧ3000P QPCR System (Stratagene,
La Jolla, CA, USA). The expression of the mRNAs was calculated using the
ΔCt method.