Quantitative RNA Analysis of Mouse Brain Injury

Following completion of all behavioral tests, mice were first euthanized with Euthasol (0.1 mL/mouse) and then transcardially perfused with 40 mL ice-cold saline. RNA samples were obtained from the ipsilateral cerebral cortex surrounding the injury site and the ipsilateral hippocampus. Total RNA was extracted from flash frozen tissue samples using a cordless motorized homogenizer with RNAse-free pellet pestles (FisherBrand) followed by the miRNeasy Mini Kit (Qiagen, Cat# 74104). Complementary DNA (cDNA) was synthesized with the Verso cDNA RT kit (Thermo Scientific, Cat# AB1453B). Both kits were used according to the manufacturer’s instructions included in the kit box. Quantitative PCR for all target RNAs (see Supplemental Table S1) was performed with the TaqMan Gene Expression assay kit (Applied Biosystems). Each sample was run in duplicates with 3 stages of 40 cycles, 2 min at 50 °C, 10 s at 95 °C (denaturing step) followed by a final transcription step of 1 min at 60 °C. Gene expression was normalized by the transcription counts of GAPDH and final relative expression levels were calculated with the 2^–ΔΔCt method [36 (link), 37 (link)].