qPCR and western blot analysis of microvascular functional molecules

Protocol: qPCR and Western Blot Analysis of Blood-Brain Barrier Proteins in APP/PS1 Mice

Study Design

Cross-sectional study comparing blood-brain barrier (BBB) molecular markers in 1-month-old APP/PS1 transgenic AD mice vs age-matched wild-type controls. Assess expression of key BBB integrity proteins in cerebral cortex microvascular fractions.

...

APP-ps1-mice" style="color:#4fc3f7;margin:1.5rem 0 0.6rem;font-size:1.15rem;font-weight:700;border-bottom:2px solid rgba(79,195,247,0.3);padding-bottom:0.3rem">Protocol: qPCR and Western Blot Analysis of Blood-Brain Barrier Proteins in APP/PS1 Mice

Study Design

Cross-sectional study comparing blood-brain barrier (BBB) molecular markers in 1-month-old APP/PS1 transgenic AD mice vs age-matched wild-type controls. Assess expression of key BBB integrity proteins in cerebral cortex microvascular fractions.

Animals and Tissue Preparation

APP/PS1 transgenic mice (n=8 per group, equal sex distribution) and WT littermates (n=8)

Euthanize by CO2 inhalation at 1 month of age

Rapidly dissect brain and place in ice-cold Hibernate-E medium

Microvascular fractionation:

• Strip meninges, mince cortex in ice-cold PBS
• Homogenize with 10 strokes in Dounce homogenizer
• Centrifuge at 3,000g for 5 min to remove debris
• Collect pellet (crude microvascular fraction)
• Percoll gradient purification to isolate microvessels

5. Flash-freeze microvascular pellets in liquid nitrogen, store at -80°C

RNA Extraction and qPCR

Extract total RNA from microvascular fractions using RNeasy Micro Kit

Assess RNA integrity (RIN > 7 required for analysis)

Reverse transcribe 500 ng RNA using SuperScript IV

qPCR reactions (LightCycler 480):

• Target genes: Cldn5, Ocln, ZO1 (tight junction), Glut1 (Slc2a1), P-gp (Abcb1a)
• Reference gene: Gapdh, β-actin
• Use SYBR Green chemistry, triplicate technical replicates
• Include no-template and no-RT controls

Western Blot Analysis

Lyse microvascular pellets in RIPA buffer + protease/phosphatase inhibitors

Quantify protein (BCA assay)

Load 20 µg protein per lane on 4-12% Bis-Tris SDS-PAGE

Transfer to PVDF membrane (wet transfer, 100V, 1 hour)

Block in 5% BSA/TBST for 1 hour at RT

Primary antibodies (overnight, 4°C):

• Anti-Claudin-5 (1:500), anti-Occludin (1:500), anti-ZO-1 (1:1000)
• Anti-Glut1 (1:1000), anti-P-gp (1:500)
• Anti-β-actin (1:5000) as loading control

7. HRP-secondary antibodies (1:10,000), 1 hour RT

Develop with ECL substrate, image on ChemiDoc

Controls

• Positive control: Adult mouse brain microvessels (known high expression)
• Negative control: Liver tissue (low BBB marker expression)
• Loading control: β-actin normalization for each sample
• Technical replicates: Triplicate qPCR, duplicate western blot lanes

Expected Outcomes

Reduced Claudin-5, Occludin, ZO-1 mRNA and protein in APP/PS1 vs WT (early BBB dysfunction)

Decreased Glut1 expression indicating impaired glucose transporter function

Potential increase in P-gp (compensatory efflux upregulation)

Sex-specific differences possible (females may show earlier BBB breakdown)

Success Criteria

• [ ] RNA yield ≥ 200 ng with RIN > 7 from microvascular fraction
• [ ] All target genes amplified with Ct < 35 in ≥ 7/8 biological replicates
• [ ] ΔΔCt method with stable reference genes (Gapdh, β-actin; CV < 0.5)
• [ ] Western blot: clear bands at expected molecular weights, no saturation
• [ ] ≥20% difference in tight junction protein expression between groups (p < 0.05)
• [ ] Effect direction consistent between mRNA and protein levels

Show more

Show more