Identification of reference genes for RT-qPCR data normalisation in aging studies

Area of Science:

• Gerontology
• Molecular Biology
• Biotechnology

Background:

• Aging alters gene expression, impacting cellular functions and increasing disease risk.
• Stable reference genes are essential for accurate gene expression analysis using RT-qPCR.
• Previous studies lack validated reference genes for diverse aging models.

Purpose of the Study:

• To evaluate the expression stability of eight commonly used reference genes.
• To identify the most stable reference gene pairs across three aging models: oncogene-induced senescence (OIS), in vitro aging, and in vivo aging.
• To provide validated reference genes for reliable RT-qPCR normalization in aging research.

Main Methods:

• Evaluated eight candidate reference genes across OIS, in vitro, and in vivo aging models.
• Utilized NormFinder and geNorm algorithms to assess gene expression stability.
• Validated selected reference genes by normalizing expression data of known aging-related genes (CDKN1A, APOD, TFRC).

Main Results:

• Identified PUM1 and TBP as the most stable pair for OIS.
• Found GUSB and PUM1 to be the most stable pair for in vitro aging.
• Determined GUSB and OAZ1 as the most stable pair for in vivo aging.
• Validated candidate genes demonstrated reliable normalization of target gene expression.

Conclusions:

• Accurate normalization is critical for robust RT-qPCR data in aging studies.
• Specific reference gene pairs (PUM1/TBP, GUSB/PUM1, GUSB/OAZ1) are recommended for OIS, in vitro, and in vivo aging, respectively.
• This study provides a valuable resource for selecting stable reference genes in future aging research.