The SH-SY5Y neuroblastoma cell line was procured from National Centre for Cell Sciences, India and maintained in (DMEM) (Sigma Aldrich, USA) and Ham 's F12 nutrient mixture (F12) (Sigma Aldrich, USA) in 1:1 ratio. The medium was supplemented with 2 mmol L-glutamine (HiMedia, India), 10% (v/v) heat inactivated fetal bovine serum (FBS) (Hyclone Laboratories, Logan, UT) and 1% antibiotics (penicillin/streptomycin). Cells were cultured at 37 ºC in a humidified CO2 ¬incubator containing 5% CO2 and the medium was changed at every 2-3 days interval. Differentiation of SH-SY5Y neuroblastoma cells into neuronal cells were carried out by supplementing 10 μM retinoic acid for 5 days (Sigma Aldrich, USA) in DMEM:Ham’s F12 (1:1 ratio) without FBS [Beske
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For each group cDNA was synthesized from 500 ng of RNA using Maxima Universal First Strand cDNA Synthesis kit (Thermo Scientific, catalogue no-K1661) and consecutively 50 ng of cDNA was used for quantitative real time PCR assay. cDNA was amplified in a thermal cycle (CFX96 PCR system, BioRadLaboretories) in 20 µl reaction mixture containing 0.5 µl cDNA template (50 ng), 12.5 µl Maxima SYBR green/fluorescent qPCR master mix (2X, Thermo Scientific), 0.3 µl forward and reverse primer (100 pmol), 6.4 µl nuclease free water. The details of primer sequences, sizes of PCR products, and the annealing temperature are listed in Table-1. PCR amplification was carried out with initial denaturation at 95 °C for 2 min followed by 40 cycles of amplification for 30 s at 94 °C, annealing for 45 s at respective primer annealing temperature and extension for 45 s at 72 °C followed by final extension at 72 °C for 10 min. The data was normalized with housekeeping gene GAPDH (internal control) and expressed was analyzed in terms of 2-∆Ct for control and treatment groups [Livak and Schmittgen,
For each group cDNA was synthesized from 500 ng of RNA using Maxima Universal First Strand cDNA Synthesis kit (Thermo Scientific, catalogue no-K1661) and consecutively 50 ng of cDNA was used for quantitative real time PCR assay. cDNA was amplified in a thermal cycle (CFX96 PCR system, BioRadLaboretories) in 20 µl reaction mixture containing 0.5 µl cDNA template (50 ng), 12.5 µl Maxima SYBR green/fluorescent qPCR master mix (2X, Thermo Scientific), 0.3 µl forward and reverse primer (100 pmol), 6.4 µl nuclease free water. The details of primer sequences, sizes of PCR products, and the annealing temperature are listed in Table-1. PCR amplification was carried out with initial denaturation at 95 °C for 2 min followed by 40 cycles of amplification for 30 s at 94 °C, annealing for 45 s at respective primer annealing temperature and extension for 45 s at 72 °C followed by final extension at 72 °C for 10 min. The data was normalized with housekeeping gene GAPDH (internal control) and expressed was analyzed in terms of 2-∆Ct for control and treatment groups [Livak and Schmittgen,