Spinal Cord RNA Extraction and Real-Time PCR Analysis

Lumbosacral part of spinal cord (3/group) was cut and used for RNA isolation with TRIzol^® reagent (Invitrogen, Carlsbad, CA, United States) according to manufacturer’s instructions. RNA concentrations were measured using spectrophotometer and RNA purity was determined by measurement of A₂₆₀/A₂₈₀ and A₂₆₀/A₂₃₀ ratios. A volume equivalent to 1 μg of RNA was used for reverse transcription with High Capacity cDNA Reverse transcription kit (Applied Biosystems, Foster City, CA, United States). cDNA was then diluted 10 times and these probes were used for real-time PCR standard protocol described previously (Jakovljevic et al., 2017 (link)) with QuantStudioTM 3 Real-Time PCR System (Applied Biosystems, Foster City, CA, United States). For negative control, cDNA template was omitted from PCR mixture. Relative expression of target gene was determined by 2^-ΔΔCt method, with Gapdh (glyceraldehide-3-phosphate dehydrogenase) as reference gene (Lavrnja et al., 2015 (link)). Quality control was routinely performed by melting curve analysis and gel electrophoresis of obtained PCR products. Primer sequences are listed in Table 1.