Quantifying BACE1 Gene Expression

Total RNA was isolated and extracted from treated cells using the 5 prime “PerfectPure RNA tissue kit” (5 Prime, Inc., Gaithersburg, MD) following manufacturer’s instructions and as described previously [36 (link)]. cDNA was obtained by reverse transcribing 1 μg of extracted RNA using an iScript cDNA synthesis kit” (BioRad, Hercules, CA). cDNA was obtained by reverse transcribing 1 μg of extracted RNA using an iScript cDNA synthesis kit” (BioRad, Hercules, CA). The quantitative Real-time RT-PCR was performed using TaqMan chemistry using “Assays-on-Demand” probes (ABI, Foster City, CA) for human BACE1 (BACE1 gene) (Hs01121195_m1) and mouse Bace1 (Bace1 gene) (Mm00478664_m1), The amplification was performed using the “StepOnePlus” PCR System (ABI, Foster City, CA). The expression of specific transcripts amplified was normalized to the expression of 18s rRNA. The data were quantified and expressed as fold-change compared to the control by using the ΔΔC_Tmethod.