cell line: um-uc-3 (atcc# crl-1749) cell type: bladder cancer cells genotype/variation: transfected with gfp-tagged rhogdi2
Growth protocol
um-uc-3 (atcc# crl-1749) bladder cancer cells were stably transfected with gfp-tagged rhogdi2, sorted for expression of gfp-rhogdi2 by fluorescence activated cell sorting as described (moissoglu et al. cancer res 2009. pmid: 19276387). RNA was isolated for microarray hybridization from cells in log phase growth.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted using an RNeasy kit, per manufacturer instructions (Qiagen, Valencia, CA, USA).
Label
biotin
Label protocol
Biotinylated cRNA were prepared according to the standard Affymetrix protocol from 6 microg total RNA.
Hybridization protocol
Following fragmentation, 10 microg of cRNA were hybridized for 16 hr at 45C on GeneChip Human Genome Array HG-U133 Plus 2.0. GeneChips were washed and stained in the Affymetrix Fluidics Station 450.
Scan protocol
GeneChips were scanned using the Affymetrix Scanner 3000.
Description
As a control for experimental samples transfected with and stably expressing a GFP-RhoGDI2 fusion transgene, UM-UC-3 cells were transfected with GFP (empty vector), selected using puromycin, and sorted by FACS as described before (Moissoglu et al. Cancer Res 2009. PMID:19276387). Two biological replicates were prepared, GFP Replicates A and B, which are paired (prepared at the same times) as biological replicates A and B, respectively, of the GFP-RhoGDI2 experimental samples.
Data processing
RMA, using its standard settings as implemented in the Bioinformatics Suite commercially available as an add on for Matlab R2009B, (The Mathworks, Natick, MA).
