Rheumatoid Arthritis Clinical Trial
Official title:
NF-KB1/IKK Epsilon Genetic Expression in Patients With Rheumatoid Arthritis
Rheumatoid arthritis (RA) is a systemic and auto-immune disorder whose primary
characteristic is the chronic inflammation of joints. The objective of this study was to
evaluate whether there was an association between the NF-KB1/IKK epsilon genetic expression
and the clinical activity in RA.
60 RA patients were included in the study, 30 with clinical activity and 30 with clinical
remission The NF-KB1/IKK epsilon genetic expression was performed by real time quantitative
Polymerase chain reaction (qPCR) through the Pfaffl method of relative quantification with
Taqman probes.
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.
Genetic expression From the leukocyte package (approximately 10 to 15 mg), it was performed
a messenger RNA (mRNA) extraction using the Magna Pure LC RNA isolation kit III (Roche) in
the Magna Pure LC 2.0 Instrument. The A260/A280 nm absorbance ratio was >1.8 (quality) and
total RNA concentration was calculated by determining absorbance at 260 nm established with
the NanoPhotometer (Implen GmbH, Germany) and the extracts were adjusted to a concentration
of 20 µg of DNA for the PCR reaction.
Subsequently, the cDNA was synthesized with the Transcriptor High Fidelity cDNA Synthesis
Kit (Roche Applied Science). It was performed a real time polymerase chain reaction (qPCR),
using the specific primers and probes for NF-KB1, IKK epsilon and 18s (as constitutive
gene), using a 7500 Fast Real Time PCR System (Applied Biosystems, Applera UK, Cheshire,
UK), mixing TaqMan® Universal PCR Master Mix and the specific probes for each gene NF-KB1
(Hs00765730-m1), IKK epsilon (Hs01063858-m1) and 18S (Hs99999901-s1), (Applied Biosystems,
Applera UK, Cheshire, UK).
According to the Ct obtained from the two different groups it was calculated the relative
units of expression through the Pfaffl relative quantification method, in which it was used
as control a healthy patient.
Statistical analysis The statistical analysis was performed through the Sigmaplot software
version 12.0. Parametric and non-parametric test were used in function to the variable
distribution: among the parametric test that were performed to compare the groups, the
Student´s T-test was included; the non-parametric tests applied were: The Mann-Whitney U
test and the Shapiro-Wilk test. Finally, a receiver operating characteristic (ROC) was used
for the analysis of the genetic expression of NF-KB1/IKK epsilon with the SPSS software
version 22.0.
;
Observational Model: Case-Only, Time Perspective: Cross-Sectional
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