Rneasy mrna kit

Cells were homogenized in RNeasy mRNA kit (Qiagen) and stored at −80 °C before total RNA extractions according to the manufacturer’s protocol. cDNA synthesis was performed using Script™cDNA Synthesis Kit (Bio-Rad). cDNA was analyzed using real-time PCR SsoAdvanced™ SYBR^® Green Supermix from Bio-Rad using appropriate primers and run on a Bio-Rad CFX96 real-time quantitative PCR (qPCR) system. Gene expression was normalized to the housekeeping gene GAPDH and calculated using the 2^−ΔCt method. Melt curve analyses were performed to ensure the specificity of qPCR product. Primer sequences can be provided on request. Values are mean ± SEM of three independent experiments, and within each experiment, triplicate samples were assessed.

Brain tissue samples and CP were homogenized in RNeasy mRNA kit (Qiagen) and total RNA extraction was performed according to the manufacturers protocol. Synthesis of cDNA was carried out using the Scrip cDNA Synthesis Kit (Bio-Rad). cDNA was analyzed using real-time PCR SsoAdvance SYBR Green Supermix from Bio-Rad using appropriate primers (sequences: fw 95 –GGTGCTGGAGAATCCAA; rev 345 –CATCCGCGAATTCATGGA) and run on a Bio-Rad CFX96 real-time quantitative PCR (qPCR) system. Reactions were performed using 0.2 μM of each primer and 1 μL of diluted cDNA (1:20) in a final volume of 10 μL. Quantification cycle threshold (Cq = 39) values per target were manually estimated. Gene expression was normalized to the housekeeping gene GAPDH and calculated using the 2^−ΔCt method. Melt curve analyses were performed to ensure the specificity of qPCR products. All assays included at least two biological replicates with two technical replicates each, positive control and a non-template control.