Study setting and participants
This matched case-control study included Iranian patients with SLE a case group and healthy people as a control group in the Rafsanjan rheumatology clinic in the southeast of Iran in the third three months of 2020. The patients fulfilled the American College of Rheumatology diagnostic criteria for SLE (14). Then 16 patients were appraised with clinical examination (there are no more patients with SLE in Rafsanjan city), and laboratory tests such as ESR, CRP, RF, Anti CCP, ANA, Anti DS DNA were performed. A rheumatologist then confirmed the results.
In this matched case-control study; all SLE patients in Rafsanjan city were enrolled as the patient group. Sixteen healthy people who had no ACR criteria were recruited from among the hospital staff of Rafsanjan. Subjects that had used anti-inflammatory drugs in the last three months or had the main symptoms of SLE in their family were excluded from the control group.
Demographic and epidemiologic data including age, sex, academic education (BSc, MSc, Ph.D.), smoking at least one cigarette a day, body mass index (BMI), and job status were matched between the two groups.
A 10ml blood sample was obtained from each subject in both groups. A sample of the blood (3ml) was reserved for ELISA assay, and an additional sample (7ml) was collected in EDTA tubes for the Real-time PCR method. Clotted blood was centrifuged for 3-5min with 3000rpm to separate the serum. The serum was kept at -20ᵒc until analyzed for ANA and CCP via ELSA kits (Germany, AESKU) according to the kit protocol.
An RNA extraction kit was applied to extract total RNA from peripheral blood samples. Extracted RNA quality was determined via electrophoresis on the ethidium bromide pretreated agarose gels. Absorption was measured at 260/280 nm by a spectrophotometer. cDNA was synthesized using a cDNA synthesis kit (Parstous, Iran) according to the manufacturer’s instructions.
5µl SYBR of green master mix (Parstous, Iran), 1µl of the generated cDNA, and 0.8µM of forward & reverse appropriate primers (Table 1) were mixed for Real-Time quantitative PCR.
The cycling program on a BIO-RAD CFX96 system (Bio-Rad Company, USA) was completed as follows: one cycle of 94 ◦C for 30 s, 45 cycles of 94 ◦C for 5 s, and 45 cycles for 34 s. This cycle was performed in triplicate, and β-actin was evaluated as a housekeeping gene. 2−ΔΔct formula was applied for the relative amounting of the PCR product. The dissociation stages, melting curves, and quantitative analyses of the data were performed using CFX manager software version 1.1.308.111 (Bio-Rad, USA).
The list of the sequence of primers used for real-time PCR in this study
DNA methyltransferase 1 (DNMT1)
Histone deacetylase 1 (HDAC1)
β-actin (housekeeping gene)
The continuous variables were expressed as the mean ± SD, and the categorical variables were presented as a percentage and frequency. Because the data showed a non-normal distribution, the Mann-Whitney test was used to compare the parameters between patients and health groups. The relations between parameters were evaluated using the Pearson correlation coefficient. All statistical analyses were performed with SPSS (version 16.0, SPSS Inc, Chicago, IL, USA). A “P-value” less than 0.05 was considered significant.
The study was conducted in accordance with the Declaration of Helsinki. Institutional Review Board approval (code: IR.RUMS.REC.1396.119) was obtained (April 2020). The present study did not interfere with the process of diagnosis and treatment of patients and all participants signed an informed consent form.