*4.7. Database Search, RNA Extraction, and Quantitative Real-Time PCR for Their mRNA Expression Levels*

To identify the potential genes involved in the pathogenesis of atherosclerosis in RA patients, we searched the NCBI Gene Expression Omnibus (GEO) database. In group 1, which includes genes associated with hyperlipidemia or CVD, we download 250 genes with the highest (>2) fold change from GSE6054 (Monocytes of patients with familial hypercholesterolemia show alterations in cholesterol metabolism) and GSE62646 (Altered gene expression pattern in peripheral blood mononuclear cells in patients with acute myocardial infarction) through the analysis tool GE02R. In group 2, which includes differentially expressed genes from RA patients compared to healthy controls, we download 250 genes with the highest (>2) fold change from GSE56649 (Expression data from active rheumatoid arthritis patients and healthy control), GSE64707 (Gene expression of human peripheral blood cells of patients with rheumatoid arthritis), and GSE93777 (Multi-omics monitoring of drug response in rheumatoid arthritis). After an integrated analysis, we identified 20 overlapping genes from both groups as target genes for examining mRNA expression using qRT-PCR. The primer sequences of the selected genes were searched from PrimerBank-MGH-PGA (https://pga.mgh.harvard.edu/primerbank/) and are listed in Table S1. Primers were designed and synthesized by Tri-I Biotech (Taipei, Taiwan).

PBMCs were isolated using the Ficoll-PaqueTM PLUS (GE Healthcare Biosciences, Uppsala, Sweden) density gradient centrifugation. Total RNAs from PBMCs were extracted by TRI Reagent (Sigma-Aldrich, Missouri, USA) according to the manufacturer's instructions. A High-Capacity cDNA Reverse Transcriptase Kit (Thermo Fisher Scientific) was used to reverse-transcribe 2 μg RNA into cDNA used for qRT-PCR analyses. The qRT-PCR reactions were performed on the CFX96 Real-time PCR system (Bio-Rad) with IQTM SYBR Green Supermix reagent (Bio-Rad). Quantitative real-time PCR using 10 ng cDNA was performed with one cycle of preincubation at 95 ◦C for 3 min, 45 cycles of amplification (95 ◦C for 15 s, 60 ◦C for 1 min), and the melt curve detection program from 55 ◦C to 95 ◦C. The difference in expression in the target gene relative to the averaged internal control gene was calculated by 2−Ct, Ct <sup>=</sup> Cttargeted genes−CtActin.

We observed a significant difference in the mRNA expression levels of 10 targeted genes between RA patients and healthy controls. Subsequently, we evaluated the difference in mRNA expression levels in the 10 candidate genes between RA patients with a high L5% and with normal L5%. The results showed a significant difference in the mRNA expression levels of one gene, *ITGAX*, between the two groups. Then, we examined the mRNA expression levels of CD11c in 93 RA patients and 41 healthy controls. The primer sequences are as follows: For CD11c (*ITGAX*), 5 -CTGCAA GGGTTTACATACACGG-3 (forward) and 5 -GAATTTTGGCGGCATCCCTAC-3 (reverse); and for the housekeeping gene, *Actin*, 5 -ATTGCCGACAGGATGCAGA-3 (forward) and 5 -GAGTACTTGCGCTCAGGAGGA-3 (reverse). To standardize mRNA expression levels of CD11c, the mRNA levels of actin were also determined in parallel for each sample. The mRNA expression levels of CD11c were calculated using the comparative threshold cycle (Ct) method and evaluated by 2−-Ct, Ct <sup>=</sup> CtCD11c–CtActin.
