Reference genes selection for quantitative real-time PCR using RankAggreg method in different tissues of Capra hircus

Area of Science:

• Animal Science
• Molecular Biology
• Biotechnology

Background:

• Accurate gene expression analysis via quantitative real-time PCR (RT-qPCR) relies on stable reference genes.
• Reference genes are assumed to have consistent expression across all sample types and experimental conditions.
• Identifying suitable reference genes is crucial for reliable RT-qPCR results in domestic goats (Capra hircus).

Purpose of the Study:

• To evaluate the expression stability of nine commonly used endogenous controls in four different goat tissues.
• To determine the most reliable reference genes for RT-qPCR normalization in Capra hircus under different dietary treatments.
• To provide recommendations for accurate RT-qPCR data normalization in goat research.

Main Methods:

• Expression stability of nine candidate reference genes (GAPDH, ACTB, 18S rRNA, RPS18, HSP-90, ALAS, HMBS, ACAC, B2M) was assessed.
• Analysis was conducted across four goat tissues: liver, visceral fat, subcutaneous fat, and longissimus muscle.
• Six different software programs and a rank aggregation method were used to determine gene expression stability.

Main Results:

• Individual gene rankings for stability varied significantly among the six software programs.
• Expression patterns of the studied genes differed notably across the four goat tissues.
• Heat shock protein 90 (HSP-90) consistently ranked among the top two most stable genes in all tissues.

Conclusions:

• HSP-90 is recommended as a reliable reference gene for RT-qPCR normalization in domestic goats.
• GAPDH, ACTB, and RPS18 demonstrated the most variable expression and are unsuitable as reference genes.
• This study provides essential guidance for accurate gene expression profiling in Capra hircus research.