Age-Invariant Genes: Multi-Tissue Identification and Characterization of Murine Reference Genes

Studies of the aging transcriptome focus on genes that change with age. But what can we learn from age-invariant genes-those that remain unchanged throughout the aging process? These genes also have a practical application: they serve as reference genes (often called housekeeping genes) in expression studies. Reference genes have mostly been identified and validated in young organisms, and no systematic investigation has been done across the lifespan. Here, we build upon a common pipeline for identifying reference genes in RNA-seq datasets to identify age-invariant genes across seventeen C57BL/6 mouse tissues (brain, lung, bone marrow, muscle, white blood cells, heart, small intestine, kidney, liver, pancreas, skin, brown, gonadal, marrow, and subcutaneous adipose tissue) spanning 1 to 21+ months of age. We identify 9 pan-tissue age-invariant genes and many tissue-specific age-invariant genes. These genes are stable across the lifespan and are validated in independent bulk RNA-seq datasets and RT-qPCR. We find age-invariant genes have shorter transcripts on average and are enriched for CpG islands. Interestingly, pathway enrichment analysis for age-invariant genes identifies an overrepresentation of molecular functions associated with some, but not all, hallmarks of aging. Thus, though hallmarks of aging typically involve changes in cell maintenance mechanisms, select genes associated with these hallmarks resist fluctuations in expression with age. Finally, our analysis concludes no classical reference gene is appropriate for aging studies in all tissues. Instead, we provide tissue-specific and pan-tissue genes for assays utilizing reference gene normalization (i.e., RT-qPCR) that can be applied to animals across the lifespan.