The fecal RNA is a novel approach to study the biological adaptations of the gastrointestinal tract (GI) of neonatal dairy calves through gene expression analysis. Our objectives in this study was to determine the potential enrichment of RNA from immune cells during isolation of fecal RNA from dairy calves, by a comparative transcriptomic profiling of genes specific for polymorphonuclear leukocytes (PMNL) and macrophages (MPO) and GI tract epithelial cells (FABP2) and cytokeratin 8 (KRT8). Fecal and blood samples were simultaneously taken from 8 neonatal Holstein calves with less than 3 wk old. The total RNA was isolated from 200 mg of feces, using a Trizol based method along with the RNeasy Plus Mini Kit (Qiagen), following the manufacturer’s instructions with some modifications. The overall RNA quantity for all fecal samples was 378.8 ± 192.3 ng/µL and purity (260/280 ratio) was 2.0 ± 0.1 determined via Nanodrop. Using the same RNA isolation method, PMNL was isolated from 100 mL of total blood, and the overall RNA quantity for all PMNL samples was 55.6 ± 32.7 ng/µL, and the purity was 1.9 ± 0.1. The standard curve was composite from all samples including cDNA from fecal and PMNL. The internal control genes used in this experiment were GOLGA5, OSBPL2, SMUG1, B2M, ACTB, GAPDH, and RPS9. Normalized gene expression data were log-transformed before statistical analysis using the Proc Mixed of SAS (SAS 9.4). The mRNA expression of KRT8 was greater (P < 0.01) in fecal RNA than in PMNL, and a trend (P = 0.08) was observed for greater expression of FABP2 in fecal RNA than PMNL. In contrast to KRT8 and FABP2, the mRNA expression of MPO was not detectable on fecal RNA, and this was reflected in a lack of amplification over the standard 40 cycles of the RT-qPCR. However, the MPO was amplified on PMNL samples. Our results indicate that RNA isolated from fecal samples contain a low amount of immune cells such as PMNL and further confirms that the signal observed in genes related to inflammation in fecal samples. However, these results need to be validated between normal and diarrhea samples.