Quantitative real-time PCR

RNA isolation

Brains were dissected into ipsilateral and contralateral hemispheres and cortex and striatal regions isolated from each hemisphere 24h after TBI and sham surgery. The samples were snap frozen in liquid nitrogen and mechanically ground. Samples were mechanically homogenised in 1mL TRIzol™ using a 21” gauge needle. One-part degassed chloroform was added to 5 parts sample in TRIzol™ solution and samples were spun at 12,000g for 15 minutes. The aqueous layer was removed and transferred to filter columns from an Illustra™ RNAspin mini isolation kit (GE Healthcare, Cat. No. 25050071). Further purification and DNase treatment of the RNA was conducted as per kit instructions. RNA was eluted in 50µL RNase-free H2O, and the eluate reapplied to the spin column again and re-spun. Concentration and purity (260/280nm and 260/230nm) of RNA was measured using a NanoDrop 1000 spectrophotometer.