Spinal Cord RNA Extraction and Real-Time qPCR

A 5 mm segment of the ipsilateral side of the spinal cord centered on the impact site was removed for extraction of RNA. Total RNA was extracted using a Trizol Reagent (Invitrogen, CA, USA) according to the manufacturers’ manual. Quantitative PCR reactions were performed using the TaqMan Fast Universal PCR Master Mix Kit (Applied Biosystems, CA, US)^{35 (link)}. The mRNA for peroxisome proliferator-activated receptor-gamma coactivator 1α (PGC-1α) was measured by real-time quantitative RTPCR using PE Applied Biosystems prism model 7700. Triplicate reactions were run for each sample for both the gene of interest and the endogenous control (glyceraldehyde-3-phosphate dehydrogenase; GAPDH). Results are presented as 2-dCt, where dCt was difference between cycle threshold (Ct) values of GAPDH and gene of interest, a calculation that compensates for loading errors. Subtraction of dCt of CD rats from dCt of KD rats gives the ddCt value that was used to calculate relative expression levels in KD animals (2-ddCt) as previously described^{35 (link)}.