Human monocytic macrophage (THP-1) cells were infected with Burkholderia pseudomallei and treated with commercial antimicrobial drug ceftazidime (10mg/ml) served as a drug control. (Replicate 2).
Biomaterial provider
THP-1 cells from (ATCC, Virginia, USA) and the clinical isolates of Burkholderia pseudomallei from Department of Microbiology (National University of Singapore, Singapore).
Treatment protocol
Group IV :- Human monocytic macrophage (THP-1) cells were infected with Burkholderia pseudomallei (A600 nm = OD 0.6, ~5 x 107 cfu/ml) and treated with commercial antimicrobial drug ceftazidime (10mg/ml) served as a drug control. (Replicate 2).
Growth protocol
Human macrophage (THP-1) cells were cultured in 72 cm2 flask in DMEM culture medium supplemented with 10% fetal bovine serum (FBS). The cells were maintained at 37°C in a humidified atmosphere of 5% CO2 and 95% air. The culture medium containing THP-1 cells were infected with with Burkholderia pseudomallei (A600 nm = OD 0.6, ~5 x 107 cfu/ml) for 24h, and treated with commercial antimicrobial drug ceftazidime (10mg/ml) served as a drug control. (Replicate 2).
Extracted molecule
total RNA
Extraction protocol
All glasswares used for RNA isolation were treated with diethyl pyrocarbonate (DEPC) to ensure RNase-free environment. They were filled with DEPC-treated water (1.0ml DEPC in 1L of sterile DD-H2O), allowed to stand overnight at 37°C and then autoclaved to eliminate residual DEPC. Total RNA was isolated from THP-1 cells infected with B. pseudomallei (n = 5 flasks for each replicate) and treated with commercial antimicrobial drug ceftazidime using RNeasy® mini kit. The RNA sample was subsequently treated with RNase-free Dnase-I at room temperature for 20min and stored at -80°C until use. The isolated RNA was subjected to several levels of testing before using for microarray hybridization. The purity and quantity of the RNA was determined by A260/A280 ratios and then A260, respectively, using Biophotometer (Eppendorf, CA, USA). RNA samples that had good OD values (1.8–2.0) were further tested on gels for the integrity of both the 28S and 18S ribosomal RNA by denaturing agarose gels and visualized with ethidium bromide under UV light.
Label
Biotin
Label protocol
cRNA was labeled with Biotin according to the manufacturer’s instructions (Enzo BioArrayTM HighYieldTM RNA Transcript Labeling Kit, Affymetrix, Santa Clara, CA).
Hybridization protocol
The preparation and processing of labeled, fragmented cRNA and hybridization cocktail for hybridization has been performed according to the manufacturer’s protocols (Affymetrix, Santa Clara, CA). Immediately after the hybridization, the hybridized probe arrays underwent an automated washing and staining protocol on an Affymetrix Fluidics Station (Replicate-2).
Scan protocol
We scanned the GeneChips and processed the signals using Affymetrix genechip scanner and expression analysis algorithm (Affymetrix, Santa Clara, CA).
Description
Total RNA was isolated from THP-1 cells infected with B. pseudomallei (n = 5 flasks for each replicate) and treated with standared antimicrobial drug ceftazidime using RNeasy® mini kit. The target chip hybridization followed successful Genechip Test3 arrays hybridization. All relevant procedures, such as amplification of cDNA, fragmentation of cRNA for hybridization, and concurrent assessment of expression levels of genes were carried out. Briefly, we synthesized double-stranded cDNA from the total RNA isolated from the THP-1 cells infected with with B. pseudomallei, and treated with CAZ. We generated biotin-labeled cRNAs by in-vitro transcription from the ds-cDNA. The cRNAs were fragmented before hybridization, and a hybridization cocktail was prepared and hybridized to the oligonucleotide probes on the HG-U133A GeneChip arrays for 16h incubation at 45°C. Immediately after the hybridization, the hybridized probe arrays underwent an automated washing and staining protocol on an Affymetrix Fluidics Station. We scanned the GeneChips and processed the signals using Affymetrix genechip scanner and expression analysis algorithm (Affymetrix, Santa Clara, CA).
Data processing
Three chips were used for each experiment (Group) according to MIAME guidelines. Expression values for each gene were calculated using the Affymetrix Microarray suite (MAS v5.0) software. Using the 50th percentile of each chip’s (per chip normalization) intensity range, expression values were normalized across the sample set by scaling the average of the intensities of all genes to constant target intensity of 800 and a comparison of signal intensities using Microsoft® Excel showed results from duplicate chips highly correlated with R values = 0.96. These data were further categorized and analyzed using stringent filter (statistical inclination) in order to identify the target genes. Hence, the resultant data were further put into marketable GeneSpring v7.0 (Silicon Genetics, Redwood City, CA, USA http://www.silicongenetics.com/cgi/SiG.cgi/Products/ GeneSpring?index.smf) for temporal sequence analyses by parametric test based on cross gene error model (PCGEM). Relative expression data for each probe set were generated by normalization over the median of the entire experiment set (per gene normalization).
Submission date
Apr 23, 2007
Last update date
Jun 29, 2009
Contact name
Pachiappan Arjunan
Organization name
NUS, NIH, GRU
Department
Anatomy, Unit on Retinal Vascular Neurobiology, BMB
