7 protocols
VALUE = Normalized value from GeneSpring
The arrays were scanned at an emission wavelength of 570 nm at 2.5-mm resolution in the GCS3000 laser scanner (Affymetrix). The intensity of the hybridization for each probe pair was computed by GCOS 1.1 software
The expression value (average difference) for each gene was determined by calculating the average of differences in intensity (perfect match intensity – mismatch intensity) between its probe pairs. The expression analysis file created from each sample (chip) was imported into GeneSpring 7.2 (Agilent Technologies, Inc., Palo Alto, CA) for further data characterization. A new experiment was generated after importing data from the same organ in which data were normalized by the array to the 50th percentile of all measurements on that array. Lists of genes that were either induced or suppressed more that two fold between samples from non-infected vs. 6 days infected mice of same genotype were created by filtration-on-fold function within the presented list. The differential expression was evaluated in grouped samples from each mouse genotype (Wt or Knockout mice) and time point (non-infected vs 6 days post infection) using ANOVA. Values correspond to fold increase or suppression (threshold of two fold) of genes between infected Vs non-infected groups of same genotype.
Chips were hybridized at 45-C for 16 h and washed with fluidics protocol EukGE-WS2v5 according to Affymetrix’s recommendation
Double-stranded cDNA was synthesized from total RNA and labeled using the ENZO BioArray RNA transcript labeling kit (Enzo Life Sciences, Inc., Farmingdale, NY, USA) to generate biotinylated cRNA. Biotin-labeled cRNA was purified and fragmented randomly according to Affymetrix’s protocol. A total of 200 µL of sample cocktail containing 15 µg of fragmented and biotin-labeled cRNA was loaded onto each chip
Spleen from C57BL/6 or knockout mice non-infected or after 6 days post infection with 10E5 P. chabaudi infected red blood cells were harvested and immediately transferred into tubes containing TRIzol® reagent (Invitrogen, Carlsbad CA) and freeze in liquid nitrogen. Sample homogeinization and total RNA isolation were subsequently processed according to the manufacturer’s protocol (Invitrogen). RNA pellets were resuspended in nuclease-free water (Invitrogen) and quantified by ultraviolet spectroscopy for purity and concentration (NanoDrop, Wilmington, DE). Integrity of RNA samples was evaluated by electrophoresis in 1% agarose gel and were assessed further for RNA integrity on the Agilent Bioanalyzer (Santa Clara, CA). Equivalent aliquots of 2.5 µg of total RNA from 12 independent samples (3 non-infected C57BL/6, or MyD88-/- and 3 P. chabaudi infected C57BL/6, or MyD88-/- mice) were assessed using the Affymetrix MOE 430 2.0 GeneChip array GeneChips.
Spleen from C57BL/6 or MyD88 knockout mice non-infected or after 6 days post infection with 10E5 P. chabaudi infected red blood