|
| Status |
Public on Sep 01, 2016 |
| Title |
siRNA-Veh-r1 |
| Sample type |
RNA |
| |
|
| Source name |
T47D cells transfected with 17β-HSD1 siRNAs and treated with vehicle
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: T47D
|
| Treatment protocol |
The sense and antisense sequences of three 17β-HSD1 siRNAs were selected and synthesized as previously described6 Scramble siRNA was used as control siRNA. Two days before transfection, T47D cells in T75 flasks were cultured in steroid deprived medium (dextran-coated charcoal-treated medium). On the transfection day, cells in T75 flasks were trypsined and ressuspended in a fresh charcoal-treated medium. 3x105 cells were then reverse-transfected in 6-well plates with 200 nM mixed 17β-HSD1 specific siRNAs or with negative control siRNA using Lipofectamine siRNAMax (Invitrogen), and cells were incubated. Two days (48 hours) after transfection, cell culture media were replaced by fresh charcoal-treated medium containing either the steroid E2 (1 nM) or ethanol as a vehicle control, and cells were incubated for two more days before RNA extraction. The RNA samples included two independent biological replicates, coming from two independent cell culture experiments, for a total of eight RNA samples.
|
| Growth protocol |
T47D cells were obtained from the American Type Culture Collection (ATCC) and were propagated in phenol red-free DME high glucose medium containing 7.5 mg/l bovine insulin (Sigma, Oakville, Ontario, Canada) and 10% fetal bovine serum (Invitrogen). When indicated, FBS was treated overnight at 4oC with 2% dextran-coated charcoal to remove the remaining steroids present in the serum. Cells were incubated at 37oC in a humidified atmosphere of 95% air and 5% CO2.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated from T47D cells using Trizol Reagent (Invitogen) in 6-well plates. RNA samples were treated with DNase1, purified using RNeasy Mini Kit column (Qiagen, Mississauga, ON, Canada), and RNA quality was assessed on the Agilent Bioanalyser 2100. Analysis using the RNA 6000 Nano Chip showed good qualities for all RNA samples with the RNA Integrity Numbers (RIN) greater than 8/10 for all the samples. RNA samples for subsequent microarray analyses and reverse transcription quantitative real-time PCR (RT-qPCR) comprised of eight samples composed of two biological repetitions for each of the four treatment conditions.
|
| Label |
biotin
|
| Label protocol |
RNA samples were processed according to the manufacturer’s recommended procedures on GeneChip Whole Transcript (WT) Sense Target Labeling Assay from Affymetrix (http://www.affymetrix.com/support/downloads/manuals/wt_sensetarget_label_ manual.pdf). The assay was started with 0.2 µg of each T47D cells RNA samples and the protocol is based on the principle of performing one cycle of cDNA synthesis and in vitro transcription (IVT) for target amplification to generate cRNA following by reverse transcription reactions to synthesis the WT cDNA. About 2.7 µg sample of fragmented cDNAs was used to hybridize human oligonucleotide array Gene 1.0 ST (Genechip; Affymetrix)
|
| |
|
| Hybridization protocol |
Following fragmentation, 10 ug of cRNA were hybridized for 16 hr at 45C on GeneChip Drosophila Genome Array. GeneChips were washed and stained in the Affymetrix Fluidics Station 400.
|
| Scan protocol |
Chips were scanned with a GeneChip scanner 3000 7G (Affymetrix) and images were extracted with the GeneChip operating software (Affymetrix GCOS v1.4). The microarray processing was performed at the DNA Biochip Platform service at CHUQ-CHUL Research Centre (Québec, Canada)
|
| Data processing |
Quantified Affymetrix image files (“.CEL” files) for each of the treatment conditions (including two independent replicates per treatment condition) were exported into the statistical software environment R where the microarray analyses were performed using the Bioconductor package OneChannelGUI
|
| |
|
| Submission date |
Jan 28, 2016 |
| Last update date |
Sep 01, 2016 |
| Contact name |
Ezequiel L Calvo |
| E-mail(s) |
cezequiel@yahoo.com
|
| Organization name |
CRCHUL
|
| Department |
Molecular Endocrinilogy
|
| Lab |
Microarrays
|
| Street address |
2705 Boul. Laurier
|
| City |
Quebec |
| State/province |
Quebec |
| ZIP/Postal code |
G1V 4G2 |
| Country |
Canada |
| |
|
| Platform ID |
GPL6244 |
| Series (1) |
| GSE77345 |
Genomic analyses of breast cancer cells: 17β-hydroxysteroid dehydrogenase type 1 induces transcriptional changes in estradiol-dependent and independent manners |
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