ID_REF = VALUE = Signal intensity accepted from the probeset ABS_CALL = Probeset signal: absent or present DETECTION P-VALUE = Probeset signal: p-value (shows the coherency of the results)
MAS 5.0 Expression Analysis Defalut Setting
Following affymetrix protocol. After hybridization, the chips were stained and washed in a Genechip Fluidics Station 450(Affymetrix) and scanned by using a Genechip Array scanner 3000 7G (Affymetrix).
Following affymetrix protocol. Fragmented end-labeled cDNA was hybridized to the GeneChip® P. aeruginosa Genome arrays for 16 hours at 50 ℃ and 60 rpm as described in the Gene Chip Whole Transcript (WT) Sense Target Labeling Assay Manual (Affymetrix).
Per RNAsample, 300 ng were used as input into the Affymetrix procedure as recommended by protocol (http://www.affymetrix.com). Briefly, 300 ng of total RNA from each sample was converted to double-strand cDNA Using a random hexamer incorporating a T7 promoter, amplified RNA( cRNA) was generated from the double-stranded cDNA template though an IVT( in-vitro transcription) reaction and purified with the Affymetrix sample cleanup module. cDNA was regenerated through a random-primed reverse transcription using a dNTP mix containing dUTP. The cDNA was then fragmented by UDG and APE 1 restriction endonucleases and end-labeled by terminal transferase reaction incorporating a biotinylated dideoxynucleotide.
To lyse the cells, 1.0 mL RLT buffer (Qiagen, Inc., Valencia, CA) and 0.2 mL 0.1 mm zirconia/silica beads (Biospec) were added to the cell pellets. The tubes were closed tightly and vortexed for 50 seconds. The total RNA was isolated by following the protocol of the RNeasy Mini Kit (Qiagen) including an on-column DNase digestion with RNase-free DNase I (Qiagen). RNA quality was assessed by Agilent 2100 bioanalyser using the RNA 6000 Nano Chip (Agilent Technologies, Amstelveen, The Netherlands), and quantity was determined by ND-1000 Spectrophotometer (NanoDrop Technologies, Inc., DE, USA).