|
| Status |
Public on Sep 03, 2018 |
| Title |
SC-SP_F3DTCtrl_rep1 |
| Sample type |
RNA |
| |
|
| Source name |
SC-SP from the pituitary
|
| Organism |
Mus musculus |
| Characteristics |
gender: Female
genotype: -/iDTR
sorted population: non-sca1high
dt treatment: 3DT
|
| Treatment protocol |
-/iDTR (ctrl) and GHCre/iDTR female mice were treated with DT for 3 (3DT) or 10 (10DT) days (4 ng / g body weight, twice a day for the indicated number of days).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Mice were euthanized by exposure to CO2 and the neurointermediate lobe of the pituitary was carefully removed. The AP was isolated and dissociated into single cells using trypsin. Cells were resuspended in pituitary-optimized serum-free defined medium (SFDM; Invitrogen). Dissociated AP cells were incubated with Hoechst33342 (2.5µg/ml; Sigma-Aldrich), stained with phycoerythrin (PE)-conjugated rat anti-mouse stem cell antigen-1 (Sca1; Table 1) (BD Biosciences), incubated with propidium iodide (2µg/ml; Sigma-Aldrich) and analyzed for side population (SP) by FACS (FACSVantage or FACS Aria III; BD Biosciences). To confirm the SP phenotype, verapamil was added (100µM; Sigma-Aldrich) which blocks the SP’s dye efflux. The SP fraction not expressing Sca1 at a high level (non-Sca1high SP) has been identified as the stem cell-clustering population of the pituitary, referred to as the ‘stem cell-SP’ or SC-SP. The SC-SP and the bulk Hoechstmid+high ‘main population’ (MP) were sorted by FACS after 3DT and 10DT treatment into cold lysis solution of the RNeasy Micro kit (Qiagen, Venlo, The Netherlands). Total RNA was extracted immediately after cell collection according to the manufacturer’s protocol, typically starting from 25000-50000 cells per sort. RNA samples were stored at -80°C until further processing for whole-genome microarray
|
| Label |
Cy3
|
| Label protocol |
RNA concentration and purity were determined spectrophotometrically using the Nanodrop ND-1000 (Nanodrop Technologies) and RNA integrity was assessed using a Bioanalyser 2100 (Agilent). Per sample, an amount of 1ng of total RNA was amplified and converted to single-stranded antisense cDNA using the NuGEN Ovation Pico WTA System v2.0. All steps were carried out according to the manufacturers protocol (NuGEN).
|
| |
|
| Hybridization protocol |
2µg of purified, amplified cDNA were used as input into the Agilent Genomic DNA Enzymatic labeling according to the manufacturers instructions, without the restriction digestion step and then labeled with cyanine 3-dUTP (Cy3). The Cy3-labeled cDNA was quantitated and dye incorporation determined by Nanodrop. 3µg labeled cDNA was hybridized on Mouse Whole Genome v2 Gene Expression 4x44K MicroArray according to the One-Color microarray-Based Gene Expression Analysis User Manual, without fragmentation step.
|
| Scan protocol |
To assess the raw probe signal intensities, arrays were scanned using the Agilent DNA MicroArray Scanner with surescan High-Resolution Technology and probe signals were quantified using Agilent’s Feature Extraction software (version 10.7.3.1).
|
| Description |
Gene expression in control (non-injured) female mouse pituitary after 3DT
|
| Data processing |
Data was base 2 log-transformed and a quantile normalization was applied. Control probes and probes below background were removed.
|
| |
|
| Submission date |
Sep 14, 2015 |
| Last update date |
Sep 03, 2018 |
| Contact name |
Rekin's Janky |
| E-mail(s) |
Nucleomics.Bioinformatics@vib.be
|
| Organization name |
VIB
|
| Department |
Nucleomics Core
|
| Street address |
Herestraat 49 Box 816
|
| City |
Leuven |
| ZIP/Postal code |
B-3000 |
| Country |
Belgium |
| |
|
| Platform ID |
GPL11202 |
| Series (1) |
| GSE72992 |
Regeneration in the pituitary after cell-ablation injury: time-related aspects and molecular analysis |
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