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.