Optimisation of reference genes for gene-expression analysis in a rabbit model of left ventricular diastolic...

Area of Science:

• Molecular Biology
• Cardiovascular Research
• Gene Expression Analysis

Background:

• Left ventricular diastolic dysfunction (LVDD) involves impaired heart relaxation or increased stiffness.
• The molecular mechanisms driving LVDD are not fully understood.
• Accurate gene expression analysis is crucial for studying LVDD pathogenesis.

Purpose of the Study:

• To identify reliable normalization genes for quantitative real-time PCR (RT-PCR) in a rabbit model of LVDD.
• To validate a new rabbit model for LVDD research using molecular markers.

Main Methods:

• A hypercholesterolemic rabbit model was established by feeding a cholesterol-enriched diet.
• Echocardiography assessed diastolic function to confirm LVDD.
• RT-PCR analyzed the expression stability of 10 candidate reference genes (Gapdh, Hprt1, Ppia, Sdha, Rpl5, Actb, Eef1e1, Ywhaz, Pgk1, G6pd) using geNorm and Normfinder algorithms.

Main Results:

• Sdha, Gapdh, and Hprt1 demonstrated the highest gene expression stability according to geNorm analysis.
• Hprt1 and Rpl5 were identified as the optimal gene combination for normalization by Normfinder.
• Increased mRNA expression of Bnp, Anp, Mcp-1, and Nox-2 was observed in hypercholesterolemic rabbits, correlating with LVDD parameters.

Conclusions:

• Sdha, Gapdh, and Hprt1 are recommended as stable reference genes for RT-PCR normalization in rabbit LVDD models.
• The identified molecular markers (Bnp, Anp, Mcp-1, Nox-2) validate the established rabbit model for LVDD research.
• This study provides essential tools for advancing the molecular understanding of LVDD.