The exfoliated fecal transcriptome (exfoliome) and its resemblance to the intestinal gene expression in dairy calves

Tissue biopsies and postmortem analysis are limitations to evaluate molecular markers at tissue-level in the gastrointestinal (GI) tract. Inflammatory condition and barrier function of the GI through a non-invasive approach such as fecal RNA gene expression analysis has been reported in dairy calves. To further explore this approach, we performed a comparative transcriptome analysis via RNA-seq in GI epithelial cells in comparison to the exfoliated fecal transcriptome (exfoliome) in dairy calves. Postmortem jejunum and fecal samples (n = 6/tissue) were collected from healthy Jersey calves (5 wk old) and frozen in liquid nitrogen until total RNA isolation. The TruSeq stranded Ribo-zero rRNA reduced library was applied to the fecal RNA samples before RNA seq, targeting 2 × 50 PE reads, using the Illumina, NovaSeq (S4) platform at the University of Minnesota Genomics Center. The sequencing generated 41.6 ± 9.0 and 36.8 ± 5.6 M paired end reads in fecal and jejunum samples, respectively. Processed reads were aligned to the bovine genome using HISAT2. Differentially expressed gene (DEG) analysis was performed using a quasi-likelihood test in the EdgeR R-package applying a False Discovery Rate (FDR) \u3c 0.05 correction. Although a greater paired end reads were generated with fecal samples than jejunum, a greater (35 ± 9.0 vs 7.1 ± 3.4) number of unmapped reads were obtained in fecal samples than jejunum. A linear discriminatory analysis was used to confirm the congruency between exfoliome and jejunum transcriptomes. Among those DEGs, the water channel protein AQP8 was upregulated in feces compared with jejunum. Cell membrane transporter genes (i.e., SLC27A5 and SLC16A6), and genes involved in the metabolism of proteins and cell proliferation (i.e., CHST4, B4GALNT3, and FGFBP1) were upregulated in the fecal RNA. Further analyses are needed, including the commonalities (overlap) between fecal and jejunum transcripts, and enrichment pathway analysis, to confirm that fecal RNA can reflect the intestinal transcriptome