|
| Status |
Public on Sep 21, 2017 |
| Title |
bt474_144h_PBS_treatment_rep2 |
| Sample type |
RNA |
| |
|
| Source name |
BT474_PBS_144h
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: BT474
cell type: HER2+ breast cancer cell line
receptor: ER+, HER2+, PR+
treated with: PBS for 144 hrs
|
| Treatment protocol |
SKBR3 and BT474 cells were plated at a starting density of 2 x 106 in 100 mm cell culture plates. BT474 and SKBR3 cells were treated with 6 µg/mL trastuzumab and/or PBS as control within two biological replicates for 144 hours. The media were changed in every 72 hours.
|
| Growth protocol |
HER2+ breast cancer cell lines BT474 (HTB-20) and SKBR3 (HTB-30) were purchased from the American Type Culture Collection (ATCC). SKBR3 cells were maintained in Mc Coy’s 5A medium (Lonza) with L-glutamine containing 10% fetal bovine serum (FBS), 1% penicillin-streptomycin. BT474 cells were maintained in RPMI 1640 medium with L-glutamine (Lonza) supplemented with 10% FBS, 1% penicillin-streptomycin and 2% bovine insulin. The cell lines were cultured in a humidified air supplemented with 5% CO2 at 37°C.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated with the TRIzol reagent (Invitrogen) according to the manufacturer's instructions. Absorption at 260 nm and 280 nm was measured for the determination of RNA purity.
|
| Label |
Cy3
|
| Label protocol |
Samples were then labeled with Cyanine3-pCp by using T4 RNA Ligase at 16°C for 2 hours. They were mixed with 10x blocking agent and 2x Hi-RPM hybridization buffer (Agilent Technologies), and hybridizations were performed at 55°C with rotation at 20 rpm.
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| |
|
| Hybridization protocol |
Hybridization was performed in Human miRNA Microarray, Release 19.0, 8x60K (G4870A, Agilent Technologies) platform, which was an array designed from miRBase version 19 containing 2006 miRNAs. Each miRNA sequence was represented by probes replicated at least 30 times on the array. Spike-in control solutions were first prepared and total RNA (100 ng) from each sample was dephosphorylated with calf intestine alkaline phosphatase at 37°C for 30 min. It was followed by a denaturation step that includes the incubation of samples in DMSO at 100°C for 10 minutes.
|
| Scan protocol |
After the washing step, the arrays were scanned in Roche Nimblegen instrument by using Agilent Scan Control software. The data were acquired using Agilent Feature Extraction software for miRNA microarray, generating a GeneView file that contained summarized signal intensities for each miRNA by subtracting the background after combining the intensities of replicate probes.
|
| Description |
bt474_k2
miRNA profiling after PBS treatment for 144h ; BT474 cells, rep2
|
| Data processing |
BRB Array Tools 4.3.2. stable release was used for the normalization and statistical analysis. Bioconductor packages were used for the normalization and statistical comparisons. The data were normalized by Quantile normalization method.
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| |
|
| Submission date |
Sep 20, 2017 |
| Last update date |
Jan 23, 2018 |
| Contact name |
Bala Gur Dedeoglu |
| Organization name |
Ankara University Biotechnology Institute
|
| Street address |
Dogol cad.
|
| City |
Ankara |
| ZIP/Postal code |
06110 |
| Country |
Turkey |
| |
|
| Platform ID |
GPL18044 |
| Series (1) |
| GSE104076 |
Determination of trastuzumab responsive microRNAs in SKBR3 and BT474 cells. |
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