Reference genes for gene expression analysis in proliferating and differentiating human keratinocytes

Area of Science:

• Molecular biology
• Dermatology
• Gene expression analysis

Background:

• Keratinocyte abnormalities are key in skin disorders, altering gene expression.
• Quantitative real-time RT-PCR (qRT-PCR) is vital for detecting these changes.
• Accurate gene expression requires reliable endogenous control (EC) genes, but optimal choices for normal human epidermal keratinocytes (NHEK) differentiation are unclear.

Discussion:

• This study evaluated 15 candidate EC genes for stability during in vitro calcium-induced NHEK differentiation.
• Commonly used housekeeping genes showed significant expression variability.
• YWHAZ and UBC demonstrated exceptional stability across proliferation and differentiation phases.

Key Insights:

• YWHAZ and UBC are identified as highly stable endogenous control genes for NHEK differentiation studies.
• Using multiple, validated EC genes is a best practice for accurate qRT-PCR.
• The YWHAZ/14-3-3-zeta combination is suitable for both transcript and protein level comparisons.

Outlook:

• These findings provide essential guidance for researchers studying keratinocyte biology and skin diseases.
• Standardizing reference gene selection will improve the reproducibility of gene expression studies in dermatology.
• Further validation in diverse NHEK models and disease states is warranted.