|
| Status |
Public on May 09, 2017 |
| Title |
mRNA_986_F10nM_2 |
| Sample type |
RNA |
| |
|
| Source name |
mRNA, patient 986, bisphenol F 10nM, replicate 2
|
| Organism |
Homo sapiens |
| Characteristics |
patient id: 986
cell type: Adipocyte
treatment: BPF
concentration: 10nM
|
| Treatment protocol |
They were induced to differentiate into adipocytes with either vehicle (DMSO) or the selected chemical (BPA, BPF, BPS) at 10nM or 10µM.
|
| Growth protocol |
Primary human white subcutaneous pre-adipocytes were cultured in PBM-2 medium. Confluent pre-adipocytes were induced to differentiate with PBM-2 medium supplemented with insulin, dexamethasone, IBMX and indomethacin (all supplied by Lonza) for ten days according to the instructions of the manufacturer.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Specific kits (Kit Small & Large RNA; Macherey Nagel) were used to extract all species of RNAs. The RNA concentration was determined by absorbance at 260 nm (A260), and the purity was estimated by determining the A260/A230 ratio with a Nanodrop spectrophotometer (Nanodrop Technologies, Wilmington, DE). RNA Integrity Number (RIN) was assessed with a Bioanalyzer (Agilent). This kit allowed the extraction of small and large RNAs with no degradation (RIN>9).
|
| Label |
Cy3
|
| Label protocol |
100 ng of total RNA per sample were dephosphorylated by incubation with a calf intestinal phosphatase at 37 °C for 30 min and denatured with 100% DMSO at 100 °C for 7 min. They were then labeled with Cyanine 3-pCp at 16°C for 2 hours, with a T4 ligase. Micro Bio-Spin 6 chromatography columns (Bio-Rad Laboratories, Hercules, CA) were used to purify the labeled RNA. The efficiency of the labeling procedure was verified with the Agilent miRNA Spike-in Kit.
|
| |
|
| Hybridization protocol |
For mRNA samples, a total of 600 ng of cRNA was fragmented and hybridized overnight at 65 °C. The purified labeled miRNAs were prepared with 10 × Blocking Agent and 2 × Hi-RPM hybridization buffer and incubated with the array in hybridization chambers (Agilent) for 20 hours at 55 °C. The slides were then washed as described in the protocol. The efficiency of the hybridization procedure was verified with the Agilent miRNA Spike-in Kit.
|
| Scan protocol |
Microarrays were run on a SureScan microarray scanner (Agilent Technologies). Raw data were acquired with Feature Extraction Software (ver. 10.7.3.1). Calculation of the total gene signal for each miRNA replicated probe subtracted background signals.
|
| Description |
mRNA profiling of human primary adipocytes atfer 10 days of diffenciation with 10nM of bisphenol F
|
| Data processing |
After a preliminary between-array normalization, unexpressed probes were filtered out according to detection pvalues. Expression values were normalized with housekeeping genes for mRNA and estimated using a linear model to take into account the probe affinity over the 30 probes characterizing each one of the miRNA. All analyses were performed using R (packages Limma and AgiMicroRna).
|
| |
|
| Submission date |
May 08, 2017 |
| Last update date |
Jan 23, 2018 |
| Contact name |
Odile Poulain-Godefroy |
| Organization name |
Université Lille, CNRS, INSERM, CHU Lille, Institut Pasteur de Lille
|
| Department |
Center for Infection and Immunity of Lille (CIIL)
|
| Lab |
UMR8204
|
| Street address |
1, rue du Pr Calmette
|
| City |
Lille |
| ZIP/Postal code |
59000 |
| Country |
France |
| |
|
| Platform ID |
GPL16699 |
| Series (2) |
| GSE98680 |
mRNA/miRNA expression profiles of human primary adipocytes exposed to low doses of bisphenols A, F and S [mRNA] |
| GSE98682 |
mRNA/miRNA expression profiles of human primary adipocytes exposed to low doses of bisphenols A, F and S |
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