Validation of suitable internal control genes for expression studies in aging.

Area of Science:

• Molecular Biology
• Genomics
• Immunology

Background:

• Gene expression normalization commonly relies on housekeeping genes, assuming stable expression.
• This assumption is increasingly challenged by evidence of variable expression in specific biological contexts.
• Reference gene validation in aging research is underdeveloped, lacking defined standards.

Purpose of the Study:

• To assess the expression stability of commonly used housekeeping genes in human peripheral blood mononuclear cells (PBMCs) across an aging spectrum.
• To identify reliable reference genes for accurate gene expression analysis in aging studies.

Main Methods:

• Quantitative reverse transcription polymerase chain reaction (RT-qPCR) was employed.
• Expression stability of five housekeeping genes (18s rRNA, ACTB, GAPDH, HPRT1, GUSB) was analyzed in PBMCs from donors aged 35-74 years.
• Stability was quantified using coefficient of variation, geNorm, and NormFinder algorithms.

Main Results:

• Glucuronidase (GUSB) exhibited the highest expression stability, identifying it as the most suitable reference gene.
• 18s ribosomal RNA (rRNA) demonstrated the least stability, making it unsuitable for normalization in this context.
• Aging was identified as a significant factor influencing the stability of 18s rRNA, hypoxanthine phosphoribosyltransferase 1 (HPRT1), and actin beta (ACTB) expression.

Conclusions:

• GUSB is recommended as a reliable reference gene for normalizing gene expression in human PBMCs, particularly in aging studies.
• 18s rRNA is not a suitable housekeeping gene for this cell type and age range.
• Researchers must consider age as a critical variable when selecting reference genes for gene expression studies in PBMCs.