Gene expression validation in apoE-/- mice

PRIMARY OUTCOME

Validation of high expression levels of C1QA, C1QC, and SPI1

EXPECTED OUTCOMES

## Primary Outcomes **Complement Gene Upregulation**: WD-fed apoE-/- mice show C1QA upregulation ≥3.5-fold (p < 0.001), C1QC upregulation ≥2.8-fold (p < 0.001), and SPI1 upregulation ≥2.0-fold (p < 0.01) vs. WT NC controls. This confirms activation of complement-dependent inflammation in atherosclerotic plaques. **Protein Confirmation**: C1QA and C1QC protein levels in liver homogenate correlate strongly with mRNA expression (Pearson's r ≥ 0.75, p < 0.001), confirming post-transcriptional regulation is minimal. ## Secondary Outcomes **Macrophage Localization**: C1QA+ immunofluorescence co-localizes with CD68+ macrophages (80-85% of C1QA+ cells are CD68+), confirming hepatic macrophage source of complement production. SPI1 (PU.1) shows nuclear localization consistent with transcription factor function. **Time Course**: 8-week WD timepoint shows intermediate upregulation (40-60% of 16-week levels), indicating progressive complement activation with lesion development.

SUCCESS CRITERIA

## Primary Success Criteria **Gene Expression Changes**: Top complement genes (C1QA, C1QC, SPI1) must show ≥2.0-fold upregulation in WD apoE-/- vs. WT NC with p < 0.01 (two-way ANOVA with Bonferroni post-hoc, n≥4 biological replicates per group). **Consistency**: ≥80% of replicates within each group must show directionally consistent change (same direction as group mean), confirming biological rather than technical variation. ## Secondary Success Criteria **Protein Correlation**: ELISA-based protein quantification must correlate with qRT-PCR fold change (Pearson's r ≥ 0.70), confirming transcript changes translate to protein level changes. **Histological Validation**: C1QA immunofluorescence intensity in liver sections must reflect tissue-level expression changes, supporting imaging-based readouts for future studies.

PROTOCOL

# Gene Expression Validation in apoE-/- Mice Protocol ## Phase 1: apoE-/- Mouse Breeding and Experimental Groups (Days 1-28) **Mouse Maintenance and Diet**: Obtain apoE-/- mice (C57BL/6J-Apoe<tm1Unc>/J, JAX #002052) and C57BL/6J wild-type controls. House on either: (a) normal chow (NC, 4% fat, Protocol #5VO5), or (b) Western diet (WD, 21% milk fat, 0.2% cholesterol, Harlan Teklad #TD.88137) for 8 or 16 weeks. Randomize at weaning (3 weeks). **Experimental Groups**: (a) WT + NC (n=10), (b) WT + WD (n=10), (c) apoE-/- + NC (n=12), (d) apoE-/- + WD (n=15). Match groups by sex distribution. Record body weight and food intake weekly. **Tissue Collection**: Fast mice 4 hours, collect plasma (EDTA, via cardiac puncture under isoflurane anesthesia). Perfuse with PBS. Dissect liver, aorta (arch to iliac bifurcation), spleen, and mesenteric adipose. Snap-freeze in liquid nitrogen or fix in 4% PFA. ## Phase 2: RNA Extraction and Gene Expression Profiling (Days 29-42) **RNA Extraction from Liver**: Homogenize 50 mg liver in Trizol (1 mL, 30 sec, bead disruptor). Extract total RNA per manufacturer protocol. Assess quality via Bioanalyzer (RIN ≥ 8.0 for all samples). Treat with DNase I (DNA-free kit). Verify purity (A260/280 ≥ 1.9, A260/230 ≥ 1.8). **qRT-PCR Array for Complement/Inflammation Genes**: Run TaqMan Array Mouse Cardioid 96-well plates (Applied Biosystems #4413277) covering 84 genes: complement system (C1QA, C1QB, C1QC, C3, C5), inflammatory cytokines (IL1B, IL6, TNF, CCL2), endothelial function (CDH5, KLF2, NOS3), lipid metabolism (LDLR, APOE, ABCA1). Include housekeepers (GAPDH, HPRT1). **Individual Gene Validation**: Validate top differentially expressed targets via individual TaqMan assays (C1QA #Mm00432142_m1, C1QC #Mm01150960_g1, SPI1 #Mm00453882_m1). Run on QuantStudio 7 Flex. Calculate fold change via ΔΔCt method (2^-ΔΔCt). Normalize to HPRT1. ## Phase 3: Protein and Histological Validation (Days 43-56) **Complement Protein Measurement**: Measure C1QA and C1QC protein in liver homogenates via ELISA (C1QA: Abcam #ab200007, C1QC: Cusabio #CSB-EL004562MO). Normalize to total protein (BCA assay). Run in duplicate with standard curve. **Immunofluorescence Staining**: Fix frozen liver sections (8 μm) in acetone (-20°C, 10 min). Block with 5% normal donkey serum. Stain C1QA (1:100, Abcam #ab90442), SPI1 (PU.1, 1:100, Abcam #ab184935), and CD68 (macrophage marker, 1:200, Abcam #ab783). Detect with Alexa Fluor 488/594 secondaries. Image on Zeiss LSM 880 confocal (20×). **Morphometric Analysis**: Quantify C1QA+ area fraction in liver sections using ImageJ. Correlate with gene expression (qRT-PCR) and protein levels (ELISA) via Pearson correlation.

LINKED HYPOTHESES

HTML iframe

<iframe src="http://scidex.ai/artifact/exp-80202e28-afd9-4db9-bc00-69a863627e48?embed=1" width="100%" height="600" style="border:0;border-radius:8px"></iframe>

Markdown link

[Gene expression validation in apoE-/- mice](http://scidex.ai/artifact/exp-80202e28-afd9-4db9-bc00-69a863627e48)

Direct URL

http://scidex.ai/artifact/exp-80202e28-afd9-4db9-bc00-69a863627e48

Preview