Computational protocol: DNA microarray of global transcription factor mutant reveals membrane-related proteins involved in n-butanol tolerance in Escherichia coli

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Protocol publication

[…] Escherichia coli strains harboring σ70 mutant B8 and WT were cultured overnight and inoculated (1 %) into fresh medium. n-Butanol (0.8 %, v/v) was added at 0.8 OD660 and further incubated for 1.5 h. Cells were harvested by centrifugation (8800 g, 4 °C). Total RNA was extracted using Qiagen RNeasy kit (Hilden, Germany) following the manufacturer’s instructions. Qualified total RNA was further purified by Qiagen RNeasy mini kit and RNase-Free DNase Set. Three biological replicates of RNA samples were stored in dry ice and subjected to further DNA microarray analysis. The microarray service was provided by Shanghai Biotechnology Co., Ltd. (Shanghai, China) using Agilent SurePrint E. coli 8 × 15 K slides, and the quality and integrity of RNA were examined before analysis.Slides were scanned by Agilent Microarray Scanner (Santa Clara, CA, USA), and data were extracted with Agilent Feature Extraction software 10.7. Raw data were normalized by Quantile algorithm, Gene Spring Software 11.0 (Santa Clara, CA, USA). Differentially expressed genes were identified using the rank product method []. The MeV (TM4) software was used for clustering and other expression profile analysis []. The related metabolic pathway of differentially expressed genes was analyzed using Kyoto Encyclopedia of Gene and Genomics (KEGG) database []. The microarray data have been deposited at the gene expression omnibus (GEO) under the accession number GSE79305. […]

Pipeline specifications

Software tools Agilent Feature Extraction, TM4
Databases GEO KEGG
Application Gene expression microarray analysis
Organisms Escherichia coli
Diseases Drug-Related Side Effects and Adverse Reactions
Chemicals 1-Butanol