DNA extraction and genotyping
The study was conducted in accordance with the ethical guidelines outlined in the 1975 Declaration of Helsinki and was approved by the Ethics Committee of the Department of Biology at the University of Tabriz (14000907-198-9). Written informed consent was obtained from all participants prior to their inclusion in the study.
EDTA-whole blood samples were collected from 200 women with lupus and 200 age, sex, and ethnically-matched healthy individuals as a control group. Genomic DNA was extracted from peripheral white blood cells using a nanoparticle-based DNA extraction commercial kit manufactured by ZiAViZ company.
To genotype the rs1632947 (-964A/G) polymorphism, we utilized TaqMan probe Real-Time PCR. This involved two primers (forward 5′-CCACTCACATGTGGATACTTCCTAAA-3′ and reverse 5′-CAGTCACAATGCCCCTCACTAG-3′) and two fluorescently labeled probes (VIC-5′-AGGCAGTGCGTGAGCA-3′-MGB and FAM-5′-AGGCAGTGCATGAGCA-3′-MGB).
The genotyping of the rs1233334(-725 C/G) polymorphism was performed using a Real-Time PCR System with two primers (forward 5′-ACT GTC TGG GAA AGTGAAACTTAAGAG-3′ and reverse 5′-AATGTGACTTTGGCCTGTTGGTATA-3′) and two fluorescently labeled probes (5′-VIC-CTT TGT GAG TCG TGT TGTA-NFQ-3′ and 5′-FAM-CTTTGTGAGTCCTGTTGTA-NFQ-3′).
The PCR conditions were as follows: an initial denaturation step at 95°C for 10 min, followed by 40 cycles of denaturation at 95°C for 15 s and annealing/extension at 60°C for 1 min. The genotyping was confirmed by direct sequencing.
The reaction mixture consisted of approximately 20 ng of genomic DNA, 1x TaqMan Universal PCR Master Mix (without AmpErase Uracil N-Glycosylase), primers, and probes, with a total volume of 10 µl. The PCR conditions were as follows: an initial denaturation step at 95∘C for 10 min, followed by 40 cycles of denaturation at 95∘C for 15 s and annealing/extension at 60∘C for 1 min. The plates were read and analyzed on a 7300 Real-Time PCR System (Applied Biosystems).
The genotyping of the 14 bp deletion/insertion polymorphism (rs371194629) located in the 3' untranslated region (3' UTR) of the HLA-G gene was performed by polymerase chain reaction (PCR) with primers flanking the 14-nucleotide InDel polymorphism. The sequences of the primers used for amplification were: Forward primer: 5’-GTGATGGGCTGTTTAAAGTGTCACC-3’ and Reverse primer: 5’-GGAAGGAATGCAGTTCAGCATGA-3’. The PCR reaction was performed in a final volume of 25 µl containing 0.2 mM of each dNTP, 2.5 mM MgCl2, 2.5 µl of 10X PCR Buffer (pH 8.5), 0.1 µM of both primers, 1 Unit of Taq polymerase, and 2 µl of template DNA per reaction. The PCR cycling conditions were as follows: an initial denaturation at 94°C for 3 min, followed by 30 cycles of 20 sec at 94°C, 20 sec at 58°C, and 30 sec at 72°C, and ending with a final extension of 3 min at 72°C in a PCR System thermal cycler. The PCR reaction for the insertion type allele product was a 224bp amplicon, and the allele containing the 14 bp deletion polymorphism was 210bp. The PCR products were analyzed by electrophoresis in a 15% polyacrylamide gel and stained with ethidium bromide to determine the presence or absence of the 14 bp deletion/insertion polymorphism.
RNA extraction and cDNA synthesize
Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood using density gradient centrifugation with sodium citrate as an anticoagulant. The PBMCs were washed with phosphate-buffered saline, and RNA was extracted using TRIzol. The extracted RNA was then used to synthesize cDNA for qPCR analysis. To obtain the cDNA needed for expression assay, we performed a reverse transcription PCR (RT-PCR) assay. First, a mixture was created by combining 5 µl of RNA (measured by nanodrop), 1 µl of oligo(dT) primer, and 5 µl of distilled water. The mixture was heated at 70℃ for 5 minutes and then rapidly cooled on ice. Next, 6.5 µl of PCR master mix was added, and the final mixture was heated to 42℃ for 1 hour. The reaction was terminated by heating at 70℃ for 5 minutes in a thermocycler.
Real-time assay (qPCR)
The expression levels of HLA-G genes in the study group and healthy controls were assessed using the SYBR Green I-based qPCR technique on a StepOnePlus™ Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). The qPCR assays were performed in triplicate using AccuPower® 2X GreenStar™ qPCR Master Mix (Bioneer, Daejon, South Korea) with specific primers (5-CACGCACAGACTGACAGAATG-3 and 5-GCCATCGTAGGCATACTGTTCA-3). PCR reactions included 100 ng cDNA, 12.5 µL of 2X GreenStar™ qPCR Master Mix, and 2 µl of each primer pair (10 pmol/µL), in a final volume of 25 µl. The PCR cycling reactions were initiated with an initial denaturation step at 94°C for 5 min, followed by 40 cycles of denaturation at 94°C for 30 sec, annealing at temperatures given in Table 1 for each primer pair for 30 sec, and extension at 72°C for 30 sec. The mRNA level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control to normalize the expression of HLA-G genes. The GAPDH primer sequences were as follows: 5-GAAAGGTGAAGGTCGGAGTC-3 and 5-GAAGATGGTGATGGGATTTC-3.
Measurement of sHLA-G Levels
The concentration of sHLA-G (IU/mL) in plasma was measured using a sandwich ELISA kit (Exbio/Biovendor, Czech Republic), according to the manufacturer's protocol. The standard curve was established by measuring the concentration of HLA-G1 and sHLA-G5 isoforms ranging from 3.91 to 125 IU/mL. The limit of sHLA-G detection in this test was 0.6 IU/mL. Samples with sHLA-G concentrations exceeding 125 IU/mL were retested after diluting them by a factor of 1:4.
Statistical analyses
Statistical analyses and graph generation were performed using GraphPad Prism software (v8.04). A p-value of 0.05 was considered statistically significant throughout the study.