4.3. Microarrays and Global Gene Expression

To assess the overall transcriptome in the disease process, we conducted a global gene expression array analysis in a small subgroup of eight participants selected from the total study population, consisting of four individuals with MASLD and four individuals in the control group. The decision to work with a small subgroup was driven by the difficulty in identifying multiple participants with closely aligned experimental characteristics within such a compact cohort during the recruitment phase. In order to assess the initial screening of genes showing differential expression, we carefully selected the best-matched combination of participants, ensuring that variables such as age, sex, BMI, HbA1c, and other significant demographic and clinical factors were balanced across both groups (Table 1B for details). Microarray analysis was performed using the Clarion S Assay, Affymetrix (Cat. # 902927, Santa Clara, CA, USA), with 24,000 transcripts. Differentially expressed gene sets were analyzed from the microarray results using a one-way ANOVA model by Partek Genomic Suites (GS- V.4.1; Partek Inc., St. Louis, MI, USA). Probe summarization and probe set normalization were performed using the GC-RMA algorithm, which included GC-RMA background correction, quantile normalization, log2 transformation, and median polish probe set summarization. The study outcomes maintained a false positive percentage <5% and a significance level.