Brain RNA Isolation and qRT-PCR Analysis

To extract total RNA, an aliquot of brain homogenate was mixed with RNABee (Amsbio) reagent, and the solution was incubated on ice with chloroform. The resultant aqueous layer was removed, and RNA was isolated using PureLink RNA Mini Columns (Life Technologies) according to manufacturer’s protocol. Contaminating genomic DNA was removed, and complementary DNA (cDNA) was reverse-transcribed with High Capacity RNA to cDNA conversion kit (Life Technologies) from equal concentrations of RNA across samples. Pre-amplification (14 cycles) was subsequently performed using TaqMan PreAmp Master Mix (Applied Biosystems) with desired targets. Pre-amplified, diluted cDNA was then used to run quantitative, real-time RT-PCR on a StepOne Plus Real-Time PCR System with desired TaqMan probes (all from Applied Biosystems; probe numbers are available upon request). Expression levels of mRNA were determined relative to housekeeping genes GAPDH and 18S RNA, and data were normalized to a wildtype calibrator sample. Statistics were performed at the relative (delta C_t) level [19 (link)].