|
| Status |
Public on Aug 29, 2008 |
| Title |
RBP2 siRNA, biol. repl. 3 |
| Sample type |
RNA |
| |
|
| Source name |
transfected SAOS-2 cells
|
| Organism |
Homo sapiens |
| Characteristics |
cells with RBP2 knockdown
|
| Treatment protocol |
SAOS-2 were transfected with 1 μg of pBabePuro plasmid and 40 nM siRNA 4sc for RBP2scsiRNA samples or siRNA 4 for RBP2siRNA samples; SAOS-2 were transfected with 1 μg of pBabePuro-HA-RB(379-928) plasmid and 40 nM siRNA 4sc for RB sample. Both transfections and RNA isolations were done separately for each set of three samples (from 1 to 3).
|
| Growth protocol |
SAOS-2 cells were transfected using Lipofectamine 2000 (Invitrogen) and placed under selection with 0.6 μg/ml puromycin. Total RNA isolation was carried out on the fifth day after transfection for samples RBP2siRNA and RBP2scsiRNA and on the third day for samples RB.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted using the NucleoSpin RNA kit (Clontech) according to the manufacturer's instructions.
|
| Label |
biotin
|
| Label protocol |
RNA was converted into cDNA and second strand cDNA synthesis was then carried out. The double-stranded cDNA was used as the template in an in vitro transcription reaction containing biotinylated CTP and UTP. The biotinylated complementary RNA (cRNA) was fragmented and purified.
|
| |
|
| Hybridization protocol |
The fragmented cRNA was added to a hybridization solution containing several biotinylated controls and hybridized to the microarray overnight at 45C
|
| Scan protocol |
Each cRNA bound at its complementary oligonucleotide is excited using a confocal laser scanner, and the positions and intensities of the fluorescent emissions are captured. These measures provide the basis of subsequent biostatistical analysis. dChip computes its own Model-Based Expression Indices (MBEIs) directly from the .CEL files. We download all .CEL files into a common directory. If the .TXT files are included in the same directory, dChip will use the presence calls calculated by MAS 5.0.
|
| Description |
gene expression data from SAOS-2 cells treated with RBP2 siRNA 4
|
| Data processing |
The data were analyzed with dChip using default analysis settings. The chip with median overall brightness is chosen, and all other arrays are normalized to it. dChip software, designed by Li and Wong 2001 (Model-based analysis of oligonucleotide arrays: Expression index computation and outlier detection. Proc. Natl. Acad. Sci. Vol. 98, 31-36) provides model-based estimates of expression levels of genes
|
| |
|
| Submission date |
Nov 26, 2007 |
| Last update date |
Aug 14, 2011 |
| Contact name |
Elizaveta V Benevolenskaya |
| E-mail(s) |
evb@uic.edu
|
| Phone |
312-413-8947
|
| Organization name |
University of Illinois at Chicago
|
| Department |
Biochemistry and Molecular Genetics
|
| Street address |
900 S. Ashland Ave.
|
| City |
Chicago |
| State/province |
IL |
| ZIP/Postal code |
60607 |
| Country |
USA |
| |
|
| Platform ID |
GPL571 |
| Series (1) |
| GSE9690 |
SAOS-2 cells, RBP2 knockdown or pRB overexpression |
|
