Transcriptomic profiling and differential expression analysis

Expected Outcomes

Primary Outcomes

Expected Outcomes

Primary Outcomes

Secondary Outcomes

• Microglial activation signature (Trem2, Cx3cr1, Aif1)
• Astrocyte reactivity markers (Gfap, S100b)
• Potential early amyloid processing gene changes (App, Bace1, Psen1)

Null Result Interpretation

• At 1 month (pre-symptomatic), changes may be subtle
• May need older timepoints (3, 6 months) to capture robust transcriptome changes
• Consider cell-type specific approaches (single-cell RNA-seq)

Success Criteria

Primary

• [ ] RIN > 8.0, ≥5 µg RNA per sample, 6 biological replicates per group
• [ ] ≥30M reads/sample, Q30 > 85%, ≥80% uniquely mapped
• [ ] DESeq2: ≥100 significant DEGs (FDR < 0.05)
• [ ] GSEA: ≥3 enriched pathways (FDR < 0.05)

Secondary

• [ ] ERCC spike-in recovery within expected range
• [ ] Correlation between replicates: r > 0.9
• [ ] Sex-specific DEGs identified and reported

Success Criteria

Primary

• [ ] RIN > 8.0, ≥5 µg RNA per sample, 6 biological replicates per group
• [ ] ≥30M reads/sample, Q30 > 85%, ≥80% uniquely mapped
• [ ] DESeq2: ≥100 significant DEGs (FDR < 0.05)
• [ ] GSEA: ≥3 enriched pathways (FDR < 0.05)

Secondary

• [ ] ERCC spike-in recovery within expected range
• [ ] Correlation between replicates: r > 0.9
• [ ] Sex-specific DEGs identified and reported

Technical Quality Gates

• [ ] Library complexity: unique fragments > 80% for all libraries
• [ ] Ribosomal reads < 5% after cleaning
• [ ] Blinded differential expression analysis