Daily Longitudinal RNAseq Profiling of Rattus Norvegicus Exposed to Liver Toxic Drugs

Living organisms are intricate systems with dynamic internal processes. Their RNA, protein, and metabolite levels fluctuate in response to variations in health and environmental conditions. Among these, RNA expression is particularly accessible for comprehensive analysis, thanks to the evolution of high throughput sequencing technologies in recent years. This progress has enabled researchers to identify unique RNA patterns associated with various diseases, as well as to develop predictive and prognostic biomarkers for therapy response. Such cross-sectional studies allow for the identification of differentially expressed genes (DEGs) between groups, but they have limitations. Specifically, they often fail to capture the temporal changes in gene expression following individual perturbations and may lead to significant false discoveries due to inherent noise in RNA sequencing sample preparation and data collection. To address these challenges, our study hypothesized that frequent, longitudinal RNA sequencing (RNAseq) analysis of blood samples could offer a more profound understanding of the temporal dynamics of gene expression in response to drug interventions, while also enhancing the accuracy of identifying genes influenced by these drugs. In this research, we conducted RNAseq on 829 blood samples collected from 84 Sprague-Dawley lab rats. Excluding the control group, each rat was administered one of four different compounds known for liver toxicity: tetracycline, isoniazid, valproate, and carbon tetrachloride. We developed specialized bioinformatics tools to pinpoint genes that exhibit temporal variation in response to these treatments. Following a 3-day acclimatization period for the rats in their new environments, daily blood collection procedures commenced, with the initial day of collection designated as Day 1. A volume of 200µL was consistently drawn for each procedure. This specific volume was strategically chosen to achieve a dual objective: firstly, to gather an adequate amount of biospecimen for both analytical and storage purposes; and secondly, to mitigate any excessive physiological stress or harm to the rats resulting from the phlebotomy process. The determination of this volume took into account the potential cumulative impact of the daily blood collection routine, adhering to protocols sanctioned by the Institutional Animal Care and Use Committee (IACUC). To enhance the reliability of the experimental results and reduce the influence of extraneous variables, all blood draws were scheduled at approximately 10:00am local time each day. Pertinent to the experimental design, selected rats received an intravenous dose of the test drug on either Day 3 or Day 8, roughly 30 minutes prior to the ensuing blood draw. It should be noted that the initial blood specimen obtained post-administration may not fully encapsulate the comprehensive effects of the drug, given the unique pharmacokinetic properties of each compound under investigation. Upon the conclusion of each experimental iteration, liver necropsy samples from the rats were subjected to conventional histopathological analysis.