Aliquots of 30 mL liquid cultures were treated with RNAprotect to stabilize RNA (QIAGEN, Valencia, CA, USA) in a ratio 1 part culture to 1.6 parts reagent as outlined by the manufacturer. RNA was subsequently extracted from the cells using a GENTRA Purescript RNA isolation kit (Gentra Systems, MN, USA) according to the manufacturer’s protocol. A DNase treatment step was included after RNA extraction in which DNase I (Roche Inc., Basel, Switzerland) was added to tubes (3 U/10 µg RNA), incubated for 30 min at 37°C, and followed by enzyme inactivation at 95°C for 5 min. RNA extracts were purified with an RNeasy Mini Kit and RNase-free DNase (QIAGEN) according to the manufacturer’s protocols. RNA was finally eluted with 50 ml RNase-free water and stored at -80°C until cDNA synthesis. Aliquots were analyzed with a Bioanalyzer (Agilent, Santa Clara, CA), which indicated minimal degradation and concentrations ranging from 409 to 620 µg/ml, and A260/A280 ratios ranging from 1.8 to 2.1.
Label
Cy3
Label protocol
cDNA production and labeling were performed by NimbleGen Systems, Inc (Madison, WI). After thawing RNA samples on ice, 10 µg total RNA was used to perform cDNA synthesis with random hexamers and SuperScript II reverse transcriptase (Invitrogen, Carlsbad, CA). RNase A and H were then used to digest the RNA. The resulting single-stranded cDNA was purified by phenol extraction and precipitated after adding 10 mg glycogen (as carrier), 0.1 volume of ammonium acetate and 2.5 volumes of 100% ethanol. The resulting pellet was dried and suspended in 30 µL water and the cDNA yield was measured by UV/visible spectrophotometry at 260 nm. The cDNA was partially digested with DNAse I (0.2 U) at 37°C for approx. 13 min, generating 50- to 200-base fragments as observed with a Bioanalyzer (Agilent). The fragmented cDNA was end-labeled with Biotin-N6-ddATP and terminal deoxynucleotidyl transferase (51 U) during incubation for 2 hr at 37°C. The labeled product was concentrated to 20 µL final volume using a Microcon YM-10 10,000 MWCO filter device (Millipore, Billerica, MA) and stored at -20 °C before hybridization.
Hybridization protocol
NimbleGen Systems, Inc. Hybriwheel technology was used to perform array hybridization. Briefly, arrays were pre-hybridized at 45°C in 50 mM MES (4-Morpholineethanesulfonic acid) buffer with 500 mM NaCl, 10 mM EDTA, and 0.005% Tween-20. Herring sperm DNA was added at 0.1 mg/ml to prevent non-specific binding. After 15 min of pre-hybridization, 4 mg of labeled cDNA in hybridization buffer was added to arrays followed by incubation for 16-20 hr at 45°C. Free probe was removed by conducting several wash steps, progressing from less to more stringent conditions. Bound probe was detected with Cy3-labeled streptavidin with signal amplification achieved by adding biotinylated anti-streptavidin goat antibody.
Scan protocol
The arrays were analyzed using an Axon GenePix 4000B Scanner (Molecular Devices Corp., Sunnyvale, CA). ImageJ software was used to rotate images and double their size without interpolation. Features were extracted using GenePix 3.0 software, using a fixed feature size. The log-transformed signal (base 2) was used as the input data for analysis.
Description
CHIP_ID: 20392, 20397
Data processing
Data analysis was performed using the R statistical package and tools available from the Bioconductor project. Data was quantile-normalized, and background corrected and summarized using the Robust Multi-array Average (RMA) method. A linear model was fitted for each gene to estimate log-ratios between multiple target RNA samples simultaneously using the LIMMA package. The standard errors of the estimated log-fold changes were moderated using empirical Bayes methods implemented in the LIMMA package, generating a moderated t-statistic. P-values were obtained from this moderated t-statistic, after adjusting for multiple hypothesis testing using Benjamini and Hochberg’s method to control the false discovery rate. We defined significant up- or downregulation as expression fold change ³2 and p-value <0.05 (actual p-values for this group were <0.01).
