Triplicate 15 ml cultures containing synchronous, ring stage parasites at 5 % parasitaemia were treated with 600 nM antimycin A in DMSO (or 1/2000 vol. DMSO for controls) for 24 hours.
Growth protocol
Blood stage P. falciparum 3D7 was maintained in continuous culture in vitro at 37 ºC using the candle-jar method. Cultures were maintained in citrated human blood at 2 % hematocrit with complete culture media (RPMI 1640 + L-Glutamine (Gibco) with 5 % w/v AlbuMAX I (Invitrogen), 50 μg.ml-1 hypoxanthine, 25 mM HEPES (Gibco) and 25 μg.ml-1 gentamycin (Sigma), pH of 7.6). Parasitaemia was assessed by thin blood smears stained with Giemsa (Sigma). Percentage infection of red blood cells was determined by cell counts under an oil immersion light microscope. Synchronous parasites were obtained by the Stockholm sorbitol method on two consecutive life cycles
Extracted molecule
total RNA
Extraction protocol
RNA was harvested using Trizol (Invitrogen) as described (Kyes, 2004). RNA concentration and purity were determined using a NanoDrop (Thermo) and RNA integrity was tested using an Agilent Bioanalyzer (Cambridge Genomic Services, Department of Pathology)
Label
Biotin
Label protocol
Microarray cRNA synthesis, hybridisation and plate-reading were conducted by Geneservice (Nottingham, UK).
Hybridization protocol
Microarray cRNA synthesis, hybridisation and plate-reading were conducted by Geneservice (Nottingham, UK).
Scan protocol
Microarray cRNA synthesis, hybridisation and plate-reading were conducted by Geneservice (Nottingham, UK).
Description
Gene expression data from LD50 antimycin A-treated Plasmodium falciparum
Data processing
Raw CEL files were imported into Bioconductor (Gentleman et al., 2004) for data analysis. Microarrays were analysed for RNA degradation prior to normalisation. Non-Plasmodium probesets were removed using the altcdfenvs package (Gautier et al., 2004) and microarray data were normalised using GCRMA (Wu et al., 2004). Differential gene expression was analysed using Linear Models for Microarray Analysis (LIMMA) (Smyth, 2004); the triplicate antimycin A treatment microarrays were contrasted against the triplicate control (DMSO-treated) microarrays.
