|
| Status |
Public on Feb 19, 2020 |
| Title |
INS1E_25-OH VitD+22 mM glucose_rep3 |
| Sample type |
RNA |
| |
|
| Source name |
INS1E cells in RPMI with pre-treatment with 25(OH)vitaminD and subsequent glucose stimulation
|
| Organism |
Rattus norvegicus |
| Characteristics |
cell line: INS1E
cell type: beta-cell line
treatment: with pre-treatment with 25(OH)vitaminD and subsequent glucose stimulation
|
| Treatment protocol |
After four passages INS1E cells (105 cells per well) were seeded in RPMI with 10 nM 25(OH)vitamin D (Sigma-Aldrich, Netherlands), RPMI with 10 nM 1,25(OH)2vitamin D (Sigma-Aldrich, Israel) or RPMI with standard supplements alone (control cells). After 72 hours of preincubation, glucose stimulation was carried out. The cells were first incubated with glucose-free Krebs-Ringer buffer for 60 minutes, and immediately after they were subjected to stimulation with 22 mM glucose in Krebs-Ringer buffer in triplicates for 60 minutes.
|
| Growth protocol |
INS1E cells were grown in monolayer in RPMI medium (Gibco by Life technologies, Paisley, UK) at 11 mM glucose with supplements in a humidified atmosphere at 37°C with 5% CO2 (20).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from cells cultured in the absence or presence of vitamin D metabolites and stimulated with or without glucose for 60 minutes using the RNeasy Mini kit (Qiagen, Austin, USA) according to the producer’s instructions.
|
| Label |
biotin
|
| Label protocol |
150 ng of total RNA from each treatment group with three parallel samples was subjected to GeneChip HT One-Cycle cDNA Synthesis Kit and GeneChip HT IVT Labeling Kit, following the manufacturer’s protocol for whole genome gene expression analysis (Affymetrix, Santa Clara, CA, USA). Labeled and fragmented single stranded cDNAs were hybridized to GeneChip® Rat Gene 1.0 ST Arrays (Affymetrix) containing approximately 28 000 gene transcripts.
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| |
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| Hybridization protocol |
The arrays were washed and stained using FS-450 fluidics station (Affymetrix).
|
| Scan protocol |
Signal intensities were detected by Hewlett Packard Gene Array Scanner 3000 7G (Hewlett Packard, Palo Alto, CA, USA). The scanned images were processed using AGCC (AffymetrixGeneChip Command Console) software.
|
| Description |
25-OH VitD+ 22 mM glucose 3
|
| Data processing |
For statistical analysis the Affymetrix.CEL files (containing probe intensities) were imported into Partek Genomics Suite software (Partek, Inc. MO, USA). Robust microarray analysis (RMA) yielding normalized log2 transformed signal intensities were applied for normalization (http://bip.weizmann.ac.il/toolbox/overview/Partek_Users_Guide.pdf). Gene transcripts with maximal signal values of less than 5 (Log2 values) across all arrays were removed to filter for low and non-expressed genes, reducing the number of gene transcripts to 17017 (provided in the 'Microarray INS1E Normalized Signal Values Mette Eskild Bornstedt.xlsx'). Differentially expressed genes between groups were identified using one-way ANOVA as implemented in Partek Genomics Suite.
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| |
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| Submission date |
Feb 18, 2020 |
| Last update date |
Feb 19, 2020 |
| Contact name |
Mette Eskild Bornstedt |
| E-mail(s) |
metbo@icloud.com, metbo@ous-hf.no
|
| Phone |
0047 91822819
|
| Organization name |
Oslo University Hospital
|
| Department |
Dep of Medical Biochemistry
|
| Lab |
Hormone laboratory
|
| Street address |
Pb 4959 Nydalen
|
| City |
Oslo |
| ZIP/Postal code |
N-0424 |
| Country |
Norway |
| |
|
| Platform ID |
GPL6247 |
| Series (1) |
| GSE145460 |
Expression data from INS1E cells stimulated with vitamin D metabolites and glucose |
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