|
| Status |
Public on Oct 06, 2013 |
| Title |
M-BE_control_rep1 |
| Sample type |
RNA |
| |
|
| Source name |
M-BE cells, control
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: M-BE
cell type: immortalized human bronchial epithelial cells
treatment: none
treatment duration: 6 days
|
| Treatment protocol |
M-BE cells in cultures were treated with human recombinant TGF-β1 (R&D system, Minneapolis, MN, USA) at a final concentration of 5 ng/ml for six days (changing fresh medium with the same concentration of TGF-β1 every three days). M-BE cultured without TGF-β1 were considered as controls.
|
| Growth protocol |
The immortalized human bronchial epithelium cell line M-BE was cultured in serum-free LHC-9 medium, and incubated at 37 ℃ with 3.5% CO2.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was prepared using TRIzol reagent (Invitrogen, Carlsbad, CA), and then purified using the RNeasy Mini Kit (Qiagen, Germantown, MD), according to the manufacturer's instructions. RNA was quantitated using a ND-1000 UV-VIS Spectrophotometer (NanoDrop Technologies, Wilmington, DE), and the integrity of the RNA was assessed with the RNA 6000 LabChip kit in combination with the Agilent 2100 Bioanalyzer (Agilent, Pal Alto, CA).
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 0.5 ug RNA using the One-Color Low RNA Input Linear Amplification PLUS kit (Agilent) according to the manufacturer's instructions, followed by RNeasy column purification (QIAGEN, Valencia, CA). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.
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| |
|
| Hybridization protocol |
1.65 ug of Cy3-labelled cRNA (specific activity >10.0 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 55 μl containing 25x Agilent fragmentation buffer and 10x Agilent blocking agent following the manufacturer's instructions. On completion of the fragmentation reaction, 55 μl of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent Whole Human Genome Oligo Microarrays (G4112F) for 17 hours at 65°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute with 37°C GE Wash buffer 2 (Agilent), then dried immediately by brief centrifugation.
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2505B) using one color scan setting for 4x44k array slides (Scan Area 61x21.6 mm, Scan resolution 5um, Dye channel is set to Green and Green PMT is set to 100% and 10%, dynamic range is extended).
|
| Description |
Con1
Gene expression in control M-BE cells.
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software v9.1 (Agilent) using default parameters (protocol GE1_105_Dec08 and Grid: 014850_D_20070207) to obtain background subtracted and spatially detrended Processed Signal intensities. Log2-transformed raw intensities were normalized across a series of arrays with the median-absolute scaling method using the limma package of R software.
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| |
|
| Submission date |
Aug 24, 2012 |
| Last update date |
Oct 06, 2013 |
| Contact name |
Bangrong Cao |
| E-mail(s) |
caobangronghe@163.com
|
| Organization name |
Cancer Institute & Hospital, Chinese Academy of Medical Sciences
|
| Street address |
NO.17 Panjiayuannanli, Chaoyang District, Beijing, P.R.China
|
| City |
Beijing |
| ZIP/Postal code |
100021 |
| Country |
China |
| |
|
| Platform ID |
GPL6480 |
| Series (1) |
| GSE40374 |
Gene expression in an EMT model using an immortalized bronchial epithelial cell line induced by TGF-β1 |
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