|
| Status |
Public on Mar 23, 2018 |
| Title |
Mm_colon_Healthy_None_rep02 |
| Sample type |
RNA |
| |
|
| Source name |
Colon from healthy female mouse, no treatment
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6JOlaHsd
tissue: colon, middle part
age: ~11 weeks
disease: Healthy
Sex: F
treatment: None
|
| Treatment protocol |
Colitis was induced by the addition of DSS (3% DSS; TdB Consultancy AB, Uppsala Sweden, molecular mass 40 kDa) dissolved in the drinking water for 5 consecutive days. The DSS solutions were made freshly every morning. Cyclosporine (CsA) 25mg/kg (Sandimmun Neoral, Novartis Pharma GmbH, Germany from 100 mg/ml) was suspended in 2% methocel solution and administered orally daily from day 1 to day 15 in an administration volume of 10 ml/kg (according to Sann et al, 2013). Intragastric tube of probiotic mixture of 1x10e9 L930BB and 1x10e9 IM386 strains suspended in 100 μl saline solution was administered prior the addition of DSS and then simultaneously with DSS.
|
| Growth protocol |
Female C57BL/6JOlaHsd mice (Harlan, Italy) were obtained at 7 weeks of age and acclimatized for at least one week before entering the study. Mice were housed in groups of 5 mice per cage (Ehret, Germany; 825 cm2 floor space) in standard controlled environmental conditions (22±1°C room temperature and 55±10 relative humidity) with a 12-h light/dark cycle on bedding material (Lignocel ¾, Germany) at Medical experimental centre (Ljubljana, Slovenia) and received standard rodent diet (Altromin 1324, Germany) and drinking water ad libitum.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Mice were euthanized by CO2 overdosing and spleen and the entire colon (from ceacum to the anus) were removed. Each colon was measured (wet length) and then divided into three parts; the distal and proximal parts were taken for histological evaluation and the middle part was used for RNA extraction. Colon samples for RNA extraction were cut into two parts, snap-frozen in liquid nitrogen and stored at -80 °C until used. Tissue was homogenized in 1000 µl of TRI Reagent (Sigma) and total RNA was isolated according to the manufacturer's instructions. RNA quantity and quality were assessed with Agilent 2100 Bioanalyzer instrument, all samples isolated fulfilled the minimum requirements (OD260/280 and OD230/280>1.8) for microarray hybridization.
|
| Label |
biotin
|
| Label protocol |
Total RNA was used to synthesize double-stranded cDNA tagged with a T7 promoter sequence. The double-stranded cDNA was subsequently used as a template for in vitro transcription with T7 RNA polymerase producing cRNA. Single-stranded cDNA was synthesized by reverse transcription of cRNA and treated with a combination of uracil DNA glycosylase (UDG) and apurinic/apyrimidinic endonuclease 1 (APE 1) that specifically recognized the unnatural dUTP residues and broke the DNA strand. DNA was labelled by terminal deoxynucleotidyl transferase (TdT) with the Affymetrix proprietary DNA Labelling Reagent that is covalently linked to biotin.
|
| |
|
| Hybridization protocol |
100 ng of total RNA was used for hybridization of Affymetrix GeneChip® Mouse Gene 2.0 ST Arrays according to manufacturer's instructions (Manual Target Preparation for GeneChip® Whole Transcript (WT) Expression Arrays – P/N 703147 Rev. 3). Labeled samples were injected into Affymetrix arrays and hybridized at 45°C and 60 rpm for 16 h in an Affymetrix Hybridization Oven. Arrays were later stained and washed in the Affymetrix GeneChip Fluidic Station 450.
|
| Scan protocol |
Arrays were scanned using Affymetrix GeneChip Scanner 3000 7G.
|
| Description |
Gene expression data in colon from healthy female mouse, no treatment
Sal02
|
| Data processing |
GeneChip data was processed and analyzed using R/Bioconductor packages. Data were summarized (w.r.t. core meta-probeset annotations and AFFX control probes, probes grouped to the level of transcripts) and quantile-normalized (background computed from antigenomic probes) using RMA algorithm from XPS package using the following parameters: xps::rma(normalize=TRUE, option=transcript, background=antigenomic, exonlevel=metacore+affx, params = list(16384, 0.0, 1.0, 10, 0.01, 1)).
MoGene-2_0-st.pgf
MoGene-2_0-st.mps
In the data table, 'null' denotes the filtered-out values.
|
| |
|
| Submission date |
Nov 15, 2016 |
| Last update date |
Mar 24, 2018 |
| Contact name |
Peter Juvan |
| Phone |
+386 1 543 7595
|
| Organization name |
Faculty of Medicine, University of Ljubljana
|
| Department |
Institute of Biochemistry
|
| Lab |
Center for Functional Genomics and Bio-Chips
|
| Street address |
Zaloska 4
|
| City |
Ljubljana |
| ZIP/Postal code |
SI-1000 |
| Country |
Slovenia |
| |
|
| Platform ID |
GPL16570 |
| Series (1) |
| GSE89851 |
The effect of new probiotic mixture in DSS (dextran sulphate sodium) – induced colitis mouse model |
|
