tissue: distal colon strain: Balb/c treatment: 5 mg/kg bw/day of E171 gender: male time point: 7 days
Treatment protocol
Gavage of vehicle (water)
Extracted molecule
total RNA
Extraction protocol
Isolation of RNA was performed using the miRNeasy Mini Kit (Qiagen, The Netherlands) including a DNase treatment, according to the manufacturer’s protocols for “Animal Cells and Animal Tissues”
Label
Cy3
Label protocol
Total RNA was synthesized into cRNA and labelled according to the One-Color Microarray-Based Gene Expression Analysis protocol version 6.6
Hybridization protocol
Hybridization was performed according to the manufacturer’s protocol on SurePrint G3 mouse Gene exp 60kv2 microarrays slides
Scan protocol
After hybridization, the microarray slides were scanned using an Agilent DNA Microarray Scanner with Surescan High-resolution Technology (Agilent Technologies, The Netherlands) with scanner settings to Dye Channel: G, Profile: AgilentG3_GX_1Color, Scan region: Agilent HD (61 x 21.3 mm), Scan resolution 3 µm, Tiff file dynamic range: 20 bit, Red PMT gain: 100%, Green PMT gain: 100%.
Data processing
First the quality of the microarrays was checked by the quality control pipeline provided by Agilent (Feature extraction software (FES) version 10.7.3.1). All samples met the quality criteria of the FES. In order to have a thorough quality check and normalize the data, an in-house QC pipeline was developed and is publically available (github.com/BiGCAT-UM/arrayQC_Module). All samples met the in-house quality check. Raw data with expression values and genes were selected for data analysis based on flags and missing values. Height groups were defined: E171 2 days, 7 days, 14 days, 21 days for the exposed samples and control 2 days, 7 days, 14 days, 21 days for the controls. Within each group, at least four out of six or five out of seven samples had to have a pass for the spot. Also, within each group, unique identifiers passed when there were less than three missing values out of six or seven values. The unique identifiers with an average expression less than four in all of the groups were omitted. The missing values were pre-processed using the GenePattern ImputeMissingValues.KNN module v13, with standard settings. Unique identifiers were removed and identical Agilent probe identifiers were merged with Babelomics 5, using the median method for pre-processing data. Next, the data was re-annotated from Agilent probe identifiers to EntrezGene identifiers (EntrezGene IDs). The expression data for all genes with an identical EntrezGeneIDs were subsequently merged with Babelomics 5, using the median method for pre-processing data. Using an R-script, a Linear Mixed Model Analysis for Microarrays (LIMMA) (version 1.0) was performed to extract differentially expressed genes (DEG). The data of each control time point (control) was subtracted from the time-matched exposed mice to E171. The standard cut-off values of a fold-change (FC) of 1.5 and a p-value of 0.05 were used in LIMMA. The DEG for each time point were subsequently used in Consensus Pathway Database (CPDB) for an over-expression gene set analysis. All the available databases from CPDB were used (release 31, 1 sept. 2015) with a p<0.01.
