Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) of HSP70

Total RNA was isolated from spinal cord samples of each group using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA). Total RNA (1 μl) was used to synthesize cDNA using a TaqMan miRNA Reverse Transcription kit (Applied Biosystems; Thermo Fisher Scientific, Inc.), following treatment with DNase I for 10 min at 37°C. A SYBR Green PCR Master mix (Applied Biosystems; Thermo Fisher Scientific, Inc.) and Bio-Rad iQ5 Gradient Real-Time PCR system (Bio-Rad Laboratories, Inc.) were used to perform qPCR and analyze the gene expression levels of HSP70. The thermocycling conditions were as follows: 95°C for 15 min; followed by 40 cycles of 94°C for 15 sec, 60°C for 30 sec and 72°C for 30 sec. The HSP70 primers used for all RT-qPCR experiments were as follows: Forward, 5′-ACCAGGACACTGTTGAGTTC-3′; and reverse, 5′-ACTCATCTCCGAGTTCACAC-3′. GAPDH was used as the reference gene, with primers as follows: Forward, 5′-AAGGTGAAGGTCGGAGTCAA-3′; and reverse, 5′-AATGAAGGGGTCATTGATGG-3′. The qPCR data was analyzed using the 2^−ΔΔCq method (13).