Primary mouse embryonic fibroblasts were isolated as described previously (Todaro G.J. and Green H. (1963), J. Cell Biol., 17, 299-313; Brusselbach S.U. et al. (1995), Oncogene, 10, 79-86.).
Growth protocol
Cells were grown using high glucose-DMEM (Invitrogen) with 1% Penicillin/Streptomycin (Invitrogen) and 10% dialyzed FBS (cutoff 10,000Da, Sigma) to near confluency. Cells were serum-starved for 48h with 1% dialyzed FBS, and then incubated for 2h with media containing 200µM 4-thiouridine (s4U, Sigma), 1% FBS and 1µCi 3H-cytidine (20Ci/mmol, Biotrend).
Extracted molecule
total RNA
Extraction protocol
RNA was isolated according to the Qiagen RNeasy Midi protocol. RNA quality and yield were measured by the RNA6000 Nano assay (Bioanalyzer 2100, Agilent Technologies). 100µg of RNA were diluted in binding buffer and loaded onto ~150µl packaged volume of the equilibrated organomercurial matrix (Squarix Biotechnology Germany) loaded to a Small Spin Column (Qiagen). After 4h incubation under constant gentle rotation and light protection at 4°C, non-bound RNA was eliminated by sequential washing steps. Bound (thiolated) RNA was eluted using binding buffer supplemented with 20mM 2-mercaptoethanol and ethanol precipitated. Specific activity of eluted RNA was calculated via 3H-cytidine measurement on a Beckman Coulter LS6500.
Label
biotin
Label protocol
RNA was reverse-transcribed, linearly amplified as described previously (Kenzelmann M. et al. (2004), Genomics 83, 550-558.), and biotin-labelled via a T7-based strategy according to manufacturer's instructions (Affymetrix).
Hybridization protocol
Following fragmentation, 10µg of cRNA were hybridized to MU74Av2 GeneChip (Affymetrix) according to manufacturer's instructions.
Scan protocol
GeneChips were scanned using the GeneChip' Scanner 3000 7G System (Affymetrix).
Description
Gene expression data from primary serum starved MEF cells grown to near confluency.
Data processing
The data were analyzed with the SAS MicroArray Solution 1.0, using standard settings except the following: consistence within experimental groups (stimulus and labelling combination) was determined by array group correlation. Loglinear mixed models with Bonferroni corrections were fitted for values of perfect-matches, only with stimulus (10% FBS) and labelling (s4U) considered to be constant and the chip-ID as random.
