Gene Expression profiling of Salmonella typhimurium Wild Type and HRG Mutant under H2O2 Stress

Objective of the study is to find out the differentially regulated genes in Salmonella typhimurium subjected to H2O2 stress. Gene expression profiling was carried out using Agilent microarray platform. Keywords: H2O2 Stress Bacterial culture (WT & HRG) were grown till early stainary phase for 7 hrs in LB. 600 microgram of H2O2 was added to the cultures for 4hrs. Total RNA from Treated & Untreated Bacterial cultures were isolated using Trizol. S.typhimurium microarray was custom designed using multiple 60mer oligonucleotides covering all known transcripts of S.typhimurium genome. Array was designed at Genotypic Technology using Agilent Technologies e-Array platform. 1 microgram of RNA is polyadenylated using poly-A polymerase (pAp) enzyme and labeled using Agilents Lirak kit as per manufacturer recommended protocol (www.agilent.com). 1650ng of labeled Sample, mixed with appropriate amount of blocking buffer and hybridization buffer (Agilent Technologies) (Web link). The hybridization mix was applied on to the backings and hybridized to custom designed 60mer s.TYPHIMURIUM microarray using sure hyb chambers at 65degC for 16 hours. Slides were washed with gene expression wash buffer 1 and 2 (Agilent Technologies) followed by Acetonitrile.Laser detection of Cyanine 3 fluorescence is performed using a confocal scanning instrument containing tuned lasers, which excite Cyanine dyes at the appropriate wavelengths. Slides were scanned at 5 micron resolution using Agilent scanner.Automated feature extraction was done using Agilent’s Feature Extraction Software. Analysis of feature extracted data was done using Agilent’s GeneSpring GX V 7.3.1 software. Pre-processing of the replicate experiment data was done to flag the outliers. The normalization was done using GeneSpring GX using the recommended Per Chip and Per Gene Data Transformation: Set measurements less than 0.01 to 0.01, Per Chip: Normalize to 50th percentile, Per Gene: Normalize to Specific Samples (Treated Vs Control). Genes with log2 ratio of 1.8 and above in replicate experiments were considered as up regulated and 0.55 and below was considered down regulated. p-value was calculated by GeneSpring GX for each gene on the basis of replicate probes to indicate statistical significance