Title: Affymetrix CEL analysis. Description:
nucleic acid hybridization to array protocol
P-AFFY-3 GeneChip hybridization
normalization data transformation protocol
Image acquisition, quantification and data analysis were performed using Affymetrix? Command and Expression ConsoleTM Software. Data were normalized using the Robust Multi-array Average (RMA) algorithm built into Expression Console.
nucleic acid labeling protocol
Fragmented cDNA was labeled using terminal transferase and biotinylated Affymetrix GeneChip labeling reagent according to the manufacturer's instructions.
Aqueous solutions of peptides -magainin2, buforin II, pleurocidin and D-LAK120-AP13 were added at the following concentrations 125 ug/ml, 250 ug/ml, 62.5 ug/ml and 15.6 ug/ml, respective. Cells were incubated at 37 deg for 30 min and centrifuged at 5000 x g for 5 min after he challenge.
Escherichia coli NCTC 9001 cultures were grown overnight in Muller-Hinton broth at 37 deg. Once OD620 reached 1.00, bacterial suspensions were challenged with peptides
nucleic acid extraction protocol
RNA was extracted using RiboPureTM and enriched using MICROBExpressTM Bacterial mRNA Enrichment Kit after the DNA digestion step (Life Technologies, Paisley, UK) At each step the quality of RNA was assessed using Pico100 (Picodrop Ltd, Hinxton, UK). cDNA was synthesised from mRNA and purified using Qiagen MinElute PCR (Qiagen, Manchester, UK). cDNA was then fragmented.