4.4. Real-Time PCR

The expression of Na_v1.8 (SCN10A), Na_v1.9 (SCN11A), TNF-α (TNF), and COX-2 (Ptgs2) genes was evaluated by Real-Time PCR in the DRG of mice. Total RNA was extracted from the DRG (L4-L5) with 1 mL of TRIzol reagent (Life Technologies, Grand Island, NY, USA) and its concentration was determined by photometric measurements. A High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA) was used in the synthesis of cDNA from 1 µg of RNA, according to the manufacturer’s recommendations. Synthesis of cDNA and RNA expression analysis was performed by Real-Time PCR using TaqMan Gene Expression Assay (Thermo Fisher Scientific, Waltham, MA USA) for Na_v1.8 (Mm00501467_m1), Na_v1.9 (Mm00449377_m1), TNF-α (Mm00443258_m1), and COX-2 (Mm01307329_m1). A no-template control (NTC) and no-reverse transcription controls (No–RT) were included. All reactions were run in duplicate on an ABI7500 Sequence Detection System (Applied Biosystems) under standard thermal cycling conditions. The mean cycle threshold values from duplicate measurements were used to calculate the expression of the target gene. Gapdh (Mm99999915_g1) was used as a housekeeping gene. Gene expression was calculated using the formula 2^−ΔΔCt as previously described [58].