Chunk #12 — Materials and methods — RNA-seq analysis of miRNA and mRNA transcriptomic changes in eight brain regions of AUD subjects (192 Set 1 RNA samples) — Microarray analysis of miRNA and mRNA transcriptomic changes in six brain regions of AUD subjects (96 Set 2 RNA samples)
For miRNA transcriptome analysis, the Affymetrix GeneChipTM miRNA4.0 array (Affymetrix, Santa Clara, CA, USA) was used following the manufacturer’s instructions. This array was designed to detect all miRNAs in miRBase Release 20 [31]. It contains 30,424 probe sets for mature miRNAs of 203 species including 2578 human mature miRNA probe sets, 2025 human pre-miRNA probe sets, and 1996 human snoRNA and scaRNA probe sets. Briefly, over 100 ng of total RNAs were labeled with the FlashTag™ Biotin HSR Labeling Kit (ThermoFisher Scientific, Waltham, MA, USA) and hybridized to the array. The samples are washed, stained, and scanned using the Affymetrix Command Console and its associated robotics. Probe cell intensity files (or CEL files) for small noncoding RNAs (including miRNAs) were generated using the Affymetrix® GeneChip™ Command Console (AGCC) software. Small noncoding RNA CEL files were processed using the Affymetrix Expression Console (EC) software (v1.4.1) with the “MicroRNA Arrays – RMA (robust multi-array average) + DABG (detection above Background)-Human only” workflow as the default analysis for background adjustment and signal normalization as well as log2 transformation to create probe-level summarization files
