Internal normalization of nascent RNA sequencing experiments to identify age-related changes of the mouse liver transcriptome

We are interested to detect age-related transcriptional changes in the mouse liver as model system. However, such transcriptional changes are difficult to detect against the transcriptional noise increasing with age. Here, we investigated a relative strategy to improve the sensitivity of RNA-sequencing experiments. An equal amount of nuclei from rat and mouse liver was mixed, and subsequently nascent RNA (nasRNA) extracted. For normalization purposes, the rat sample was the same, while we used triplicate samples from 3- and 15-month-old mice. The reads obtained by Illumina massive-parallel sequencing were separated, and for each gene was calculated the ratio of mouse nasRNA to rat nasRNA. As such, we could identify age-related changes of the liver transcriptome that we were not able to detect previously.