|
| Status |
Public on Nov 10, 2011 |
| Title |
B104 siRBM3 rep1 |
| Sample type |
RNA |
| |
|
| Source name |
B104 clonal cell line, transfected, 48 hours
|
| Organism |
Mus musculus |
| Characteristics |
cell type: neuroblastoma
cell line: B104
knockdown: RBM3
|
| Treatment protocol |
Cell lines were transfected with an siRNA duplex against RBM3 at a ratio of 20 nM siRNA per 25 µl Lipofectamine RNAiMax (Life Technologies) to knockdown RBM3, or mock treated as a control; cells were harvested after 48 hrs.
|
| Growth protocol |
B104 cells were cultured in DMEM supplemented with 10% FBS, 200 mM L-glutamine and penicillin/streptomycin.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
The mirVana miRNA isolation kit (Life Technologies) was used to isolate miRNAs according to the manufactor's reccomendations. RNA was quantified using a NanoDrop-1000 spectrophotometer and quality was monitored with the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA).
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from total RNA using the microRNA Spike-In Kit (Agilent) according to the manufacturer's instructions, followed by RNAeasy column purification (QIAGEN, Valencia, CA). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.
|
| |
|
| Hybridization protocol |
Hybridization was done according to Agilent's specifications.
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2505B) using one color scan setting for 1x44k array slides (Scan Area 61x21.6 mm, Scan resolution 10um, Dye channel is set to Green and Green PMT is set to 100%).
|
| Description |
microRNA expression 48 hours after RBM3 knockdown by siRNA transfection
|
| Data processing |
Microarrays were analyzed with Genespring software. The Agilent Feature Extraction tool was used to obtain the signal values and detection above background for the Agilent chip. Processing of the data was performed within the Bioconductor project and the R program software (R is available as Free Software under the terms of the Free Software Foundation's GNU General Public License). The scale normalization method used was proposed by Yang et al [49, 50] and is further elaborated by Smyth and Speed [51]. Log-values were scaled to have the same median-absolute-deviation (MAD) across arrays. The 578 Human miRNAs were filtered down to 216 by excluding those miRNAs with signals not detected above background for all the samples.
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| |
|
| Submission date |
Nov 07, 2011 |
| Last update date |
Nov 10, 2011 |
| Contact name |
Julie Pilotte |
| E-mail(s) |
pilotte.julie@gmail.com
|
| Organization name |
The Scripps Research Institute
|
| Department |
Neurobiology
|
| Lab |
Peter Vanderklish
|
| Street address |
10550 North Torrey Pines Rd, Sbr-14
|
| City |
La Jolla |
| State/province |
CA |
| ZIP/Postal code |
92037 |
| Country |
USA |
| |
|
| Platform ID |
GPL8824 |
| Series (1) |
| GSE33519 |
Microarray profiling reveals broad and differential effects of RBM3 on miRNA expression. |
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