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Status |
Public on May 24, 2011 |
Title |
Empty vector A549 stable transfectants, culture 1 |
Sample type |
RNA |
|
|
Source name |
A549/vec cells
|
Organism |
Homo sapiens |
Characteristics |
cell line: A549 lung cancer cells disease: Lung adenocarcinoma transfection: Empty pLemiR plasmid
|
Treatment protocol |
Adherent cells on a 10 cm culture dish were rinsed with phosphate-buffered saline, and treated at 37 ºC for 5 minutes with 0.75 ml of 0.05% trypsin with 0.25 mM ethylene-diamine-tetraacetic acid. Phosphate-buffered saline (10 ml) was used to collect the detached cells which were then spun down at 500 g for 10 minutes at room temperature. The cell pellet was used to isolate RNA.
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Growth protocol |
Cells were cultured to 80%-95% confluence in 10 cm tissue culture plates in +DMEM medium containing 10% fetal bovine serum and 2 µg/ml puromycin, at 37 ºC with 5% carbon dioxide and 90% humidity.
|
Extracted molecule |
total RNA |
Extraction protocol |
Total RNA was isolated using Purelink™ RNA isolation kit (Invitrogen®) as per the manufacturer's recommended protocol. RNA quantity and quality was assessed on a Bioanalyzer™ 2100 instrument (Agilent®). The RNA preparations had 260 nm/280 nm absorbance ratios in the 1.9-2.0 range, 260 nm/230 nm absorbance ratios >1.8, and RNA integrity numbers >7.
|
Label |
Biotin
|
Label protocol |
Illumina® TotalPrep RNA Amplification kit (Ambion®) was used to convert 500 ng of RNA to cDNA which was then transcribed in vitro to generate biotin-labeled RNA.
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|
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Hybridization protocol |
Hybridization reagents were mixed with 750 ng of labeled RNA, and the mixture was hybridized overnight at 58 ºC to HumanRef-8 v3.0 Expression BeadChips™ array (Illumina®).
|
Scan protocol |
Following washing and staining with Cy3 dye-labeled streptavidin, BeadChips™ (Illumina®) were imaged using the BeadArray Reader (Illumina®)
|
Description |
A0-1 Biological replicate 1
|
Data processing |
Data was acquired with GenomeStudio™ software (version 2010.1, Illumina®), and then processed for background-correction, variance-stabilizing transformation, and then robust splines-normalization between arrays using the lumi Bioconductor package (version 1.14) for R language (version 2.11.1) with default settings.
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Submission date |
May 24, 2011 |
Last update date |
May 24, 2011 |
Contact name |
Santosh Kumar Patnaik |
Phone |
716-8458364
|
Organization name |
Roswell Park Comprehensive Cancer Center
|
Department |
Thoracic Surgery
|
Street address |
Elm and Carlton Streets
|
City |
Buffalo |
State/province |
NY |
ZIP/Postal code |
14263 |
Country |
USA |
|
|
Platform ID |
GPL6883 |
Series (1) |
GSE29496 |
Overexpression of the lung cancer-prognostic miR-146b microRNAs has a minimal and negative effect on the malignant phenotype of A549 lung cancer cells |
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