Real-time PCR for adenosine receptor A1 gene expression

Tissues were harvested from five animal groups: (1) Naïve, (2) four days after CFA administration, (3) same as group 2 but with acupuncture treated on the third day, (4) same as group 3 but with 0.3 mg/ml caffeine oral administration, and (5) same as group 3 but with 0.6 mg/ml caffeine oral administration. Groups 4 and 5 were chronically treated with systemic caffeine as described in the “caffeine administration” section above. Ipsilateral L3-L5 dorsal root ganglia (DRG) and tissue surrounding ST36 acupuncture point (roughly 3 × 3 × 3 mm³ of tibialis anterior and extensor muscles attached to tibia, without dermis) were dissected from the mice following perfusion fixation with RNAlater solution (Ambion, Foster City, CA) to preserve RNA integrity. The isolated tissues were submerged in RNAlater solution overnight at 4 °C and then, stored at –80 °C. Tissues were homogenized using FastPrep-24 5 G with CoolPrep Adapter (MP Biomedicals, Santa Ana, CA), which separates tibia from acupuncture point tissue samples. Total RNA was extracted with RNeasy Plus Universal Kit (Qiagen, Valencia, CA). Expression of the adenosine A1 receptor gene, Adora1, was assessed using TaqMan Gene Expression Assays (Mm01308023_m1), and the expression levels were normalized to multiple reference genes⁵⁰: glyceraldehyde-3-phosphate dehydrogenase (Gapdh), acidic ribosomal protein (Arbp) and hypoxanthine-guanine phosphoribosyl transferase (Hprt) for DRG, and 18 S ribosomal RNA (Rn18S), beta-actin (ActB), Gapdh and Hprt for Acupoint, before calculating relative expression.