Transcriptomic Analysis of Ileal Tissue

A part of the terminal ileum from each mouse was snap frozen in liquid nitrogen and stored at −80°C. RNA was isolated with the RNeasy kit (Qiagen, Valencia, CA, USA). Quantity of RNA was measured with the ND-1000 (NanoDrop Technologies, Thermo Fisher Scientific, Breda, the Netherlands) and quality of RNA was assessed with the Bioanalyzer 2100 (Agilent, Santa Clara, CA, USA). Total RNA (100 ng) was labeled utilizing the Ambion WT Expression kit (Life Technologies Ltd., Bleiswijk, the Netherlands) and the Affymetrix GeneChip WT Terminal Labeling kit (Affymetrix, Santa Clara, CA, USA). After labeling, samples were hybridized to Affymetrix GeneChip Mouse Gene 1.1 ST arrays. An Affymetrix GeneTitan Instrument was used for hybridization, washing, and scanning of the array plates. Bioconductor packages integrated in an online pipeline were used for quality control of the data (17 (link), 18 (link)). Probe sets were redefined using current genome information (19 (link)). Probes were reorganized based on the Entrez Gene database (remapped CDF v14.1.1). Robust Multiarray Analysis preprocessing algorithm available in the Bioconductor library affyPLM (20 (link)) was used to obtain normalized expression estimates from the raw intensity values.

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Fransen F., Sahasrabudhe N.M., Elderman M., Bosveld M., El Aidy S., Hugenholtz F., Borghuis T., Kousemaker B., Winkel S., van der Gaast-de Jongh C., de Jonge M.I., Boekschoten M.V., Smidt H., Schols H.A, & de Vos P. (2017). β2→1-Fructans Modulate the Immune System In Vivo in a Microbiota-Dependent and -Independent Fashion. Frontiers in Immunology, 8, 154.

Publication 2017

RNeasy kit ND-1000 Bioanalyzer 2100 Ambion WT Expression kit Affymetrix GeneChip WT Terminal Labeling kit GeneChip Mouse Gene 1.1 ST arrays

Frozen Gene Genechip Genome Hybridization Ileum Library Mouse Nitrogen

Corresponding Organization : TiFN

Other organizations : Wageningen University & Research, Radboud University Nijmegen, Radboud University Medical Center

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Variable analysis

independent variables

RNA isolation method (RNeasy kit)
Labeling method (Ambion WT Expression kit, Affymetrix GeneChip WT Terminal Labeling kit)
Hybridization, washing, and scanning (Affymetrix GeneTitan Instrument)
Bioinformatics analysis (Bioconductor packages, Robust Multiarray Analysis preprocessing)

dependent variables

Gene expression levels measured using Affymetrix GeneChip Mouse Gene 1.1 ST arrays

control variables

Part of the terminal ileum from each mouse
Storage of samples at -80°C
Quantity and quality of RNA measured (ND-1000, Bioanalyzer 2100)
Total RNA amount (100 ng) used for labeling

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