Microarray Analysis of Gene Expression

RNA concentration and purity were evaluated using a NanoDrop 2000c UV-Vis spectrophotometer (Thermo Scientific, United States). The RNA integrity number (RIN) was measured for each sample using Agilent 2100 Expert software. Samples with a RIN value of 6 and higher were further processed (Schroeder et al., 2006 (link)). The SurePrint G3 Human Gene Expression v3 8 × 60K Microarray (Agilent, United States) was utilized in this study. The arrays were processed according to the One-Color Microarray Based Gene Expression Analysis protocol v. 6.9.1. The slides were scanned using a SureScan Dx Microarray Scanner (Agilent, United States). The images obtained after scanning were analyzed using Agilent Feature Extraction software v. 12.0.3.1. The analysis included quality control metrics (filtering of outlier spots, background subtraction from features, and dye normalization) and report. An image and a detailed description of the quality control metrics are in Supplementary Excel File_1-QC Metrics.
The principal component analysis (PCA) was conducted on microarray data (submitted to GEO NCBI resources) using filtered flags [detected, not detected] (Figure 1).