Effect of fermentation and prebiotic supplementation on organs of healthy rats
Description
The data was used to investigate how milk and yogurt ingestion alone or combined with prebiotic inulin influences the colonic mucosa, liver, and bone transcriptome. A 6-week dietary intervention was conducted in healthy young growing male rats which received a diet containing: 1) milk, 2) milk supplemented with inulin (5% w/w), 3) yogurt, or 4) yogurt supplemented with inulin (5% w/w). The microarray results revealed that yogurt consumption resulted in 2195 upregulated differential expressed genes (DEGs) and 1474 downregulated DEGs in colonic mucosa as compared with milk consumption. According to GO categories and KEGG pathway analysis, tight junction-, immune system-related pathways in the colonic mucosa, and metabolic pathways in the liver were enriched with yogurt consumption. No evident differences were identified in the bone transcriptome between the diet groups. Overall, the study found that intake of fermented dairy exerts more pronounced effects on gene expression in the intestinal tissue than prebiotics supplementation. DNA microarray data (File Bone_, Gut_, and Liver_FDR_0.1 tacx) can only be reached using a software called Transcriptome Analysis Console. From a direct comparison of the Milk and Yogurt groups, 2195 upregulated DEGs and 1474 downregulated DEGs were identified in CMS (List of differential expression genes) To support the transcriptomic analyses, real-time PCR was performed on three selected genes (q-PCR result). The results showed that, although non-significant, the mRNA expression level of Arhgef18 was higher for the Yogurt group, consistent with transcriptomic analysis.
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Steps to reproduce
1. RNA extraction and transcriptome analysis Total RNA was extracted using Trizol according to the manufacturer protocol (Sigma-Aldrich, MO, USA) from approximately 10 mg colonic mucus scrubs, 15 mg liver tissue, and 25 mg bone powder, respectively. Then transcriptome profiling was performed using the Affymetrix Clariom S mouse as described elsewhere (Rasmussen, Thogersen et al. 2023). Briefly, cRNA was synthesized from 100 ng of total RNA using the GeneChip WT Plus Reagent Kit (Thermo Scientific) and processed according to the manufactures protocol (Thermo Scientific), after fragmentation and labeling the samples were loaded into arrays for a 16 hours of hybridization. Hereafter, the arrays were washed on the Fluidic Station 450s (Thermo Scientific) and scanned using the GeneChip Scanner (Thermo Scientific). The obtained data was analyzed using Transcriptome Analysis Console software 4.0.2. (Applied Biosystem) with FDR < 0.1. An online tool : G: Profiler (https://biit.cs.ut.ee/gprofiler/gost) was used for gene ontology (GO) categories and Kyoto Encyclopedia of Gene and Genomes (KEGG) pathway analyses. 2. qPCR An amount of 800 ng extracted RNA was subjected to reverse transcriptase into cDNA using the iScript kit (Bio-Rad, California, USA) according to the manufacturer protocol. To evaluate the specific content of mRNA of three selected genes in CMS, quantitative real-time PCR (q-PCR) was performed using TaqMan probes as previously described(Rasmussen, Zamaratskaia et al. 2011). Rat-specific primers and probes pairs were designed according to Rasmussen et al.(Rasmussen, Bertholdt et al. 2018). Relative mRNA content was calculated from the obtained Ct-values and normalized to the content of Eef1a1. The mRNA content of the housekeeping gene Eef1a1 was not significantly different between groups.