🧫 Experiment Protocol
ValidationAtherosclerosisC1QA, C1QC, SPI1apoE-/- miceproposed
This animal model experiment utilized apolipoprotein E knockout (apoE-/-) mice, a well-established model of atherosclerosis, to validate the expression of identified hub genes in vivo. These mice spontaneously develop atherosclerotic lesions similar to human disease when fed a normal or high-fat diet. The expression levels of C1QA, C1QC, and SPI1 were measured using quantitative real-time PCR in tissues from these atherosclerotic mice. This experiment provided crucial in vivo validation of the computational findings, demonstrating that the identified hub genes are indeed upregulated in a physiologically relevant animal model of atherosclerosis.
PRIMARY OUTCOME
Validation of high expression levels of C1QA, C1QC, and SPI1
EXPECTED OUTCOMES
## Primary Outcomes
**Hub Gene Upregulation**: In WD-fed apoE-/- mice (16 weeks), expect 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 chow controls. 4 additional hub genes show ≥1.8-fold change (p < 0.05).
**Macrophage Correlation**: C1QA immunofluorescence intensity correlates with aortic lesion area (Pearson's r = 0.72, p = 0.003) and CD68+ area (r = 0.81, p < 0.001), supporting macrophage source of complement proteins.
## Secondary Outcomes
**Temporal Induction**: WD 8-week timepoint shows intermediate upregulation (40-60% of 16-week levels), suggesting progressive complement activation with lesion progression. Standard chow apoE-/- mice show mild elevation (1.5-2.0-fold vs. WT chow).
SUCCESS CRITERIA
## Primary Success Criteria
**Statistical Significance**: Top 3 hub genes (C1QA, C1QC, SPI1) must show ≥2.0-fold upregulation in WD apoE-/- vs. WT chow controls with p < 0.01 (Student's t-test, n≥4 biological replicates per group).
**Biological Replication**: Fold-change direction must be consistent across all replicates within each group (≥80% of replicates showing same direction of change as group mean).
## Secondary Success Criteria
**Localization Confirmation**: C1QA protein must co-localize primarily with CD68+ macrophages in aortic lesions (Pearson's coefficient ≥0.65 at lesion core region), confirming cellular source.
**Pathway Enrichment**: Hub genes must map to relevant pathways (complement system, innate immune response, lipid metabolism) based on IPA pathway annotation, supporting biological relevance beyond statistical noise.
PROTOCOL
# Validation of Hub Genes in apoE-/- Atherosclerotic Mice Protocol
## Phase 1: apoE-/- Mouse Breeding and Atherosclerosis Induction (Days 1-42)
**Mouse Maintenance**: Maintain apoE-/- mice (C57BL/6J-Apoe<tm1Unc>/J, JAX #002052) on standard chow or Western diet (WD, 21% milk fat, 0.2% cholesterol) for 8 or 16 weeks. Compare with age-matched C57BL/6J WT controls. Housing: 12h light/dark cycle, ad libitum food/water.
**Dietary Groups**: (a) Standard chow 8 weeks (n=12), (b) WD 8 weeks (n=15), (c) WD 16 weeks (n=15). Randomize mice into groups at weaning (3 weeks). Record body weight weekly.
**Tissue Collection**: At endpoint, fast mice 4 hours, collect plasma (EDTA, 200 μL), dissect aorta (arch to iliac bifurcation), and collect liver, spleen, and adipose tissue. Snap-freeze in liquid nitrogen or fix in 4% PFA. Store at -80°C.
## Phase 2: Gene Expression Analysis (Days 43-56)
**RNA Extraction**: Homogenize aortic arch (50-100 mg) in Trizol using bead disruptor (3× 30s pulses). Extract total RNA, assess quality (Bioanalyzer RIN ≥ 7.0). Treat with DNase I (DNA-free kit).
**qRT-PCR Array**: Run TaqMan Array Mouse Cardioid 96-well plates (Applied Biosystems #4413277) covering 84 genes in relevant pathways (inflammatory cytokines, endothelial function, lipid metabolism). Include housekeepers (GAPDH, HPRT1). Run on QuantStudio 7 Flex (384-well format).
**Target Gene Validation**: Validate top differentially expressed genes (C1QA, C1QC, SPI1, and 4 additional candidates) via individual qRT-PCR assays (TaqMan). Normalize to HPRT1. Calculate fold change (2^-ΔΔCt) between apoE-/- WD and WT chow groups.
## Phase 3: Immunohistochemical Validation (Days 57-70)
**Aortic Root Sectioning**: Embed aorta in OCT, cut 10 μm cryosections at 3 levels (100 μm apart). Fix sections in acetone (-20°C, 10 min). Block with 5% normal donkey serum.
**Immunofluorescence**: Stain for C1QA (1:100, Abcam #ab90442), SPI1 (PU.1, 1:100, Abcam #ab184935), and macrophage marker CD68 (1:200, Abcam #ab783). Apply secondary antibodies: Alexa Fluor 488 (1:500) and Alexa Fluor 594 (1:500). Counterstain with DAPI. Image via confocal microscopy (20×, 3 fields per section).
**Quantification**: Measure C1QA+ area fraction and co-localization with CD68+ macrophages using ImageJ. Express as percentage of total aortic area or per CD68+ macrophage area.