|
| Status |
Public on Mar 07, 2012 |
| Title |
AGO2-RNA upon scramble miR reconstitution in LNCaP, biological replicate 2 |
| Sample type |
RNA |
| |
|
| Source name |
AGO2 bound RNA in prostate cancer cell line LNCaP upon scramble miR reconstitution
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: LNCaP
tissue: prostate cancer
tumor stage: early
|
| Treatment protocol |
Cells were transfected using the Nucleofector 2 Technology (Amaxa) according to the instructions. For miRNA reconstitution 2.5ug plasmid DNA for each miRNA was used. AGO2 RNA co-immunoprecipitation (AGO2 co-IP) was performed using AG02-1 11A antibody (Ascenion).
|
| Growth protocol |
Cells were grown in medium to confluency (LNCaP cells: RPMI 1640 medium with L-glutamine (PAA) (10%FCS (Biochrom), 100Units/ml penicillin, 100 µg/ml streptomycin (PAA) and 10mM HEPES buffer (Biochrom), PC-3 and Du-145 cells: DMEM/F-12 medium with L-glutamine (PAA) (10\%FCS (Biochrom), 100Units/ml penicillin, 100 µg/ml streptomycin (PAA)), and RWPE-1 cells: Keratinocyte-Serum free medium (Gibco-BRL) (5ng/ml human recombinant EGF and 0.05mg/ml bovine pituary extract (Gibco-BRL))).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Trizol extraction of AGO2 bound RNA was performed according to the manufacturer's instructions.
|
| Label |
biotin
|
| Label protocol |
2µg RNA were analyzed on Affymetrix GeneChip Human Genome U133A 2.0 Arrays according to the manufacturer`s protocol.
|
| |
|
| Hybridization protocol |
GeneChips were proceed using the Affymetrix GCS3000 7G system according to manufacturer`s protocol.
|
| Scan protocol |
GeneChips were proceed using the Affymetrix GCS3000 7G system according to manufacturer`s protocol.
|
| Description |
AGO2 bound RNA
For identifying direct targets of scramble miR a AGO2-RNA co-immunoprecipitation as described by (Beitzinger et al. 2007) was performed. AGO2-bound mRNAs were identified by using Affymetrix Genechips.
|
| Data processing |
The data were analyzed with R (version 2.9.0) and BioConductor (version 2.4). Quality assessment of arrays included exploration of distributions of probe intensities across all arrays, of average background values, and RNA quality was examined by RNA degradation plots. Diagnostic plots based on a probe level model (PLM) have been used to rule out chips with spatial artifacts (Chip pseudo-images, normalized unscaled standard error (NUSE) plot and relative log expression (RLE) plot). Background adjusted normalized data was retrieved by the GCRMA (robust multi-array average) procedure.
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| |
|
| Submission date |
Jul 16, 2010 |
| Last update date |
Mar 07, 2012 |
| Contact name |
Kristin Reiche |
| E-mail(s) |
kristin.reiche@izi.fraunhofer.de
|
| Organization name |
Fraunhofer Institute for Cell Therapy and Immunology
|
| Department |
Diagnostics
|
| Street address |
Perlickstr. 1
|
| City |
Leipzig |
| ZIP/Postal code |
04103 |
| Country |
Germany |
| |
|
| Platform ID |
GPL571 |
| Series (2) |
| GSE17362 |
miRNA expression, mRNA expression upon miRNA reconstitution, and direct mRNA target identification in prostate cancer cell lines |
| GSE22979 |
Profiling of direct mRNA targets of miR-130a, miR-203 and miR-205 in prostate cancer cell line LNCaP |
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