Ingenuity Pathway Analysis (IPA) of miR-130a Targets Protocol
Phase 1: Target Prediction and Network Construction (Days 1-3)
Data Preparation: Export miR-130a seed sequence (UUUUGAGU) and mature form from miRBase (v22). Curate 3'/5' UTR sequences from RefSeq for all human protein-coding genes (n=19,650). Run IPA miRNA Target Filter using Context+ scoring threshold (score ≥0.8) and seed-type conservation filter.
Network Generation: Build core analysis including: direct and indirect relationships, confidence filter set to "predicted" and "established", species limited to human/mouse/rat. Include upstream regulators, downstream effects, and causal networks.
...Ingenuity Pathway Analysis (IPA) of miR-130a Targets Protocol
Phase 1: Target Prediction and Network Construction (Days 1-3)
Data Preparation: Export miR-130a seed sequence (UUUUGAGU) and mature form from miRBase (v22). Curate 3'/5' UTR sequences from RefSeq for all human protein-coding genes (n=19,650). Run IPA miRNA Target Filter using Context+ scoring threshold (score ≥0.8) and seed-type conservation filter.
Network Generation: Build core analysis including: direct and indirect relationships, confidence filter set to "predicted" and "established", species limited to human/mouse/rat. Include upstream regulators, downstream effects, and causal networks.
Phase 2: Statistical Enrichment and Canonical Pathway Analysis (Days 4-5)
Pathway Enrichment: Run IPA Canonical Pathways analysis with Fisher's exact test (right-tailed), multiple testing correction via Benjamini-Hochberg (B-H FDR < 0.05). Focus on disease-relevant pathways: PI3K/AKT, MAPK, TGF-β, VEGF, and diabetes/cardiovascular disease-specific networks.
Upstream Regulator Analysis: Identify transcription factors and kinases predicted to be activated/inhibited based on downstream gene expression patterns. Calculate activation Z-scores (|Z| > 2.0 threshold).
Phase 3: Functional Network Refinement (Days 6-7)
Network Merging: Overlay significantly enriched pathways into a unified regulatory network centered on miR-130a. Filter connections by: (1) direct physical miRNA-mRNA interaction, (2) strong experimental evidence from >2 published studies, (3) relevance to cardiovascular/metabolic disease phenotypes.
Top Candidate Selection: Rank predicted miR-130a targets by: (a) composite network proximity score, (b) number of disease-relevant pathway memberships, (c) tissue-specific expression in heart/vascular/endothelium (GTEx dataset). Select top 20 candidates for downstream experimental validation planning.