7 protocols
VALUE = Signal
ABS_CALL = indicating whether the transcript was present (P), absent (A), or marginal (M)
Hybridisation, washing, staining, and scanning were done according to the Affymetrix manual using a gene chip fluidics station 450 (Affymetrix) and a gene chip scanner 3000 (Affymetrix). For hybridization of individual gene chips, 2–2.5 µg of labeled cDNA were used in a total volume of 150 µl hybridization solution, which contained 7% DMSO (Sigma-Aldrich, St. Louis, USA) in addition to the described composition. Hybridization was done overnight at 48°C.
Standard Affymetrix procedures
Signal intensities were detected and analyzed with the Affymetrix gene chip operating software version 1.2 (GCOS; Affymetrix) using the algorithms described in the Affymetrix statistical algorithms description document (http://www.affymetrix.com). Data were globally scaled to a target intensity of 500, and default statistical parameters of GCOS (α1 = 0.05, α2 = 0.065, τ =0.015, γ1H = 0.002,γ1L = 0.002, γ2H = 0.002667, γ2L = 0.002667, Perturbation1.1) were applied. Signal values from the arrays were then processed using Genespring GX 7.3 (Agilent Technologies,CA, USA). The experiment was normalized with the default settings for Affymetrix arrays, i.e., first data transformation to set measurements of below 0.01 to 0.01 followed by a per-chip normalization to the 50th percentile and a per-gene normalization to median. For statistical comparisons, the student t-test with a P value threshold of 0.01 was applied. Genes were considered as differentially expressed if the fold change (FC) was higher than 2 when comparing the different strains.
The fragmented cDNA was end-labeled with biotin-ddUTP in a final volume of 50 µl using terminal dexoynucleotidyl transferase (Promega) in combination with the gene chip labeling reagent (Affymetrix). The reaction was incubated for 75 min at 37°C and stopped by the addition of 2 µl of 0.5 M EDTA.
Cells were grown to mid-exponential phase (OD600 of 0.4-0.5). For harvesting, cultures were rapidly transferred to cold tubes containing one tenth of the culture volume of “stop solution” (10% Tris-HCl-buffered phenol [pH 8] in ethanol), centrifuged for 5 min (10,800 × g; 4°C), the supernatant was decanted, and cells were frozen in liquid nitrogen and stored at −80°C.
Total RNA was isolated using the hot phenol extraction procedure described previously (Babst et al. 1996). RNA integrity was checked by agarose gel electrophoresis. Precipitated RNA (100 µg) was treated with 20 units of RQ1 DNase I (Promega, Madison, USA) for 30 min at 37°C in a reaction volume of 200 µl. SUPERase•InTM (100 units; Ambion,Huntingdon, UK) was included in the reaction to inhibit potential RNase activity. RNA samples were cleaned up with RNeasy spin columns (Qiagen). cDNA was synthesized according to the Affymetrix antisense genome array protocol for E. coli (http://www.affymetrix.com). For reverse transcription, MMLV reverse transcriptase RNase H minus (Promega, Madison, USA) was used in the supplied reaction buffer. The resulting cDNA was spectrophotometrically quantified and fragmented according to the Affymetrix manual except that the time for fragmentation by DNase I was shortened to 3 min. For control, 200 ng of DNase I-treated cDNA were separated on a 2% agarose gel and stained with SYBR green II (Molecular Probes, Inc., Eugene, OR, USA). Ideally, the fragmented cDNA migrated in a range that corresponded to 50–200 bp of the 50-bp ladder (Fermentas International Inc., Burlington, Canada).