189 Comparative transcriptomic analysis between Total RNA and mRNA isolated from feces of neonatal dairy calves

Abstract

Abstract The fecal RNA method can be used to evaluate biological adaptations of the gastrointestinal tract of dairy calves through gene expression analysis. The process of RNA isolation from fecal samples presents several challenges, including the potential enrichment of prokaryotic (bacterial), RNA which can dilute the targeted eukaryotic RNA and consequently affect the sensitivity of the fecal RNA method to low expressed genes. Therefore, our objective in this study was to determine the differential eukaryotic RNA enrichment in total RNA vs mRNA from feces of healthy neonatal dairy calves. To test this, a comparative transcriptomic profiling of genes specific for epithelial cells, including cytokeratin 8 (KRT8) and aquaporin (AQP3), as well as inflammatory-related genes (TLR4 and IL1B) was performed in fecal samples collected from 6 pre-weaned Holstein calves. The total RNA was isolated from 200 mg of feces, using a Trizol based method along with the RNeasy Plus Mini Kit (Qiagen). Then, 45 μg of fecal total RNA was used to isolate mRNA through magnetic selection using Dynabeads® Oligo (dT)25 (Invitrogen). The standard curve was composite from all samples including cDNA from total RNA and mRNA. The internal control genes used in this experiment were B2M, ACTB, GAPDH, RPS9, and PPIA. Normalized gene expression data were log-transformed prior to statistical analysis using the Proc Mixed of SAS (SAS 9.4). The expression of KRT8 was greater (P = 0.03) in fecal mRNA than in fecal total RNA. A trend (P = 0.09) was observed for greater expression of TLR4 in fecal total RNA than in fecal mRNA. The expression of AQP3 and IL1B was not different. These preliminary data further confirms that fecal RNA method has potential to be used as a tool to evaluate gastrointestinal tract health in dairy calves, but further adjustments are needed to improve accuracy and robustness.