NF-KB1/IKK Epsilon Genetic Expression in Patients With Rheumatoid Arthritis

Patients with a confirmed diagnosis of RA were allocated in two groups based on the Disease Activity Score 28 (DAS28): a) with clinical activity and b) with clinical remission. Key exclusion criteria were patients with any other inflammatory or autoimmune disease and patients with infections. The control group was represented by healthy patients Sample size

The sample size calculation was carried out through the following formula:

n= Nz2 pq /d2(N-1) +z2 pq With N=30, Z=2.46 (99% of confidence), p=0.9, q=0.1, d=0.05 (95% of accuracy). The sample size contained 20 patients with RA.

Anthropometric measurements Weight (kg) and height (m) were calculated in a mechanical column scale (SECA). The patients were classified according to their Body mass index (BMI, weight (Kg)/ height (m)2) as a) normal weight (BMI< 24.9), b) overweight (24.9 kg/m2 <BMI<29.9 kg/m2) and c) obese (BMI> 30 kg/m2).

Lymphocyte extraction Lymphocytes from peripheral blood were extracted using the ACK Lysing Buffer® (Life technologies, Grand Island, NY) kit. Briefly, a venous blood sample in EDTA tube (BD Vacutainer®, Franklin Lakes, NJ), was centrifuged at 3500 rpm during 5-8 minutes. The resulting intermediate white colored phase was extracted and placed in an eppendorf tube; 1 mL of ACK Lysing Buffer® was added and carefully re-suspended. Once again, the suspension was centrifuged at 3500 rpm during 5-8 minutes and the supernatant was discarded. This last step was repeated until the leukocyte package was completely white. Finally, it was added 100 mcl of ACK Lysing Buffer® and frozen at -70ºC for later use.