Quantitative Real-Time PCR of DRG Tissues

Quantitative real-time PCR (qRT-PCR) was performed following a method described previously [23 (link)]. Animals were euthanized by decapitation, and the L4-L5 DRGs were harvested and placed in TRIzol. The total RNA was extracted by the TRIzol method. Total RNA was then reverse-transcribed using an oligo-dT primer and PrimeScript II RTase (TaKaRa). Each sample was run in triplicate in a 20 μL reaction with 10 μM forward and reverse primers, 10 μL of SYBR Green qPCR Super Mix (Invitrogen), and 25 ng of cDNA. Reactions were performed in the Applied Biosystems 7500 Fast Real-Time PCR System. GAPDH was used as an internal control to normalize samples. The relative ratio of ipsilateral-side mRNA levels to contralateral-side mRNA levels was quantified by the 2^−ΔΔCT method. The rat-specific primer sequences of the examined mRNA for qRT-PCR are listed in Table 1.