Identification of stable senescence-associated reference genes.

Area of Science:

• Cell Biology
• Molecular Biology
• Genomics

Background:

• Cellular senescence is a permanent cell cycle arrest crucial in aging and disease.
• Quantitative real-time PCR (qPCR) is widely used to study senescent cell hallmarks.
• Proper normalization using stable reference genes is critical for reliable qPCR data interpretation.

Purpose of the Study:

• To systematically review normalization strategies in senescent cell research.
• To identify and validate novel, stable reference genes for qPCR in senescent fibroblasts.
• To provide guidelines for selecting reliable reference genes in senescence studies.

Main Methods:

• Systematic literature review of qPCR normalization in senescence studies.
• Analysis of public RNA sequencing datasets to assess gene stability (Shapiro-Wilk test, coefficient of variation).
• Comparison of novel candidate reference genes against commonly used ones using RNAseq and qPCR data.

Main Results:

• Most studies on cellular senescence violate MIQE guidelines by using single, unvalidated reference genes.
• Several novel reference gene candidates demonstrated superior stability in senescent fibroblasts compared to classical genes.
• Strain-dependent variations in reference gene stability were observed.

Conclusions:

• Classical reference genes are often unstable in senescent cells, compromising qPCR data accuracy.
• This study proposes more reliable reference genes for senescent cell research.
• Adherence to validated normalization strategies is essential for reproducible senescence studies.