Title: Affymetrix CEL analysis. Description:
nucleic acid hybridization to array protocol
P-AFFY-3 GeneChip hybridization
C57BL/6J mice were obtained from CharlesRiver (Germany) and housed in specific pathogen free environment, with water and food ad libitum.
nucleic acid labeling protocol
cDNA was fragmented, labeled and hybridized onto Affymetrix GeneChip Mouse Gene 1.0 ST Arrays according to the Ambion WT Expression kit for Affymetrix GeneChip Whole Transcript (WT) Expression Array Protocol (P/N 4425209 Rev.B 05/2009) and GeneChip WT Terminal Labeling and Hybridization User Manual for use with the Ambion WT Expression kit (P/N 702808 Rev.6) (Santa Clara, USA).
nucleic acid extraction protocol
Total RNA was extracted using the phenol-clorophorm chloroform method (Chomczynski, P. & Sacchi, N. (1987). Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162: 156-159). Total RNA integrity and purity were assessed using the Agilent 2100 Bioanalyzer (Paolo Alto, USA) and RNA 6000 Nano LabChip kits. Only good quality RNA with no sign of contamination and degradation (RIN > 9) were used further processed.
Three months old male mice were intraperitoneally injected with 100 uL 1x PBS (LifeTechnologies) containing lipopolysaccharide (LPS, Enzo LifeSciences) or with 100 uL PBS vehicle as control. For the single treatment, animals were injected once with 4 ug LPS per gram body weight or vehicle and sacrificed in the 5th or 19th day since the start of the treatment. For the repeated treatment, 4 injections in consecutive days, with 1 ug LPS per gram body weight or vehicle were performed and the mice were sacrificed in the 5th or 19th day since the start of the treatment. Brain tissue was collected and homogenized.