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 can serve as reference 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. Age-invariant genes have shorter transcripts 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, even though hallmarks of aging typically involve change, select genes associated with these hallmarks resist age-related change. Finally, our analysis provides a list of murine tissues where classical reference genes are appropriate for application in aging studies. However, no classical reference gene is appropriate across all aging tissues. Instead, we provide novel tissue-specific and pan-tissue reference genes for assays utilizing gene normalization (RT-qPCR) that can be applied to mice across the lifespan.