Best reference genes for unbiased normalized transcript expression in normal and dystrophic human cell models of...

Area of Science:

• Biochemistry
• Molecular Biology
• Genetics

Background:

• Patient-derived cell models are crucial for studying muscle diseases like Duchenne muscular dystrophy (DMD).
• Accurate gene expression analysis using RT-qPCR requires reliable reference genes for normalization.
• Existing reference genes may not be suitable across different cell types or disease states.

Purpose of the Study:

• To identify and validate stably expressed reference genes for RT-qPCR in human myogenic cell lines.
• To ensure accurate gene expression normalization in both healthy and dystrophic muscle models.
• To support preclinical therapeutic assessments in muscular dystrophy research.

Main Methods:

• Screened 14 candidate reference genes in healthy and two DMD-derived immortalized muscle cell lines.
• Assessed gene expression stability throughout myogenic differentiation (proliferation to 11 days).
• Utilized four statistical algorithms (Bestkeeper, geNorm, Normfinder, DeltaCt) for comprehensive analysis.

Main Results:

• RPS18, UBC, and YWHAZ demonstrated high expression stability across all tested cell lines and differentiation stages.
• These genes proved reliable for normalization irrespective of myogenic cell line genotype or differentiation state.
• The identified panel provides a robust foundation for quantitative gene expression studies in DMD models.

Conclusions:

• RPS18, UBC, and YWHAZ are recommended as a validated reference gene panel for RT-qPCR in human myogenic cell lines.
• This finding facilitates accurate gene expression analysis in Duchenne muscular dystrophy research.
• The validated reference genes will enhance the reliability of preclinical therapeutic assessments using patient-derived models.