strain: Dd2 treatment group: EGTA IC50 time point: 2 hr
Treatment protocol
Synchronzied parasite cultures are splitted into proper number flasks (75ml culture/flask). Total number is double number of time points for one experiment. Half flasks are treated with one compound with the final concentration of IC50 or IC90, and half flasks are negative controls. After the treatment of different time points, parasites in each flask were harvested for total RNA isolation and microarray hybridizations.
Growth protocol
Laboratory strain 3D7 and Dd2, sorbitol synchronized. Parasites grow in a 2% suspension of purified human RBCs and RPMI 1640 media supplemented with 0.25% Albumax, 2 g/l sodium bicarbonate, 0.1 mM hypoxanthine, 25 mM HEPES (pH 7.4), and 50 μg/l gentamycin, at 37°C, 5% O2, and 6% CO2. The 3D7 parasites for microarray reference pool were mixed with 6 time-series samples (every 8hrs through a 48hr life cycle), harvested from a 6 liter biofermenter synchronized culture.
Extracted molecule
total RNA
Extraction protocol
RNA extraction, cDNA synthesis and labeling as well as microarray hybridizations of the four samples representing time points 1, 2, 3, and 4 against a reference RNA pool were carried out as described in Bozdech Z, Llinas M, Pulliam BL, Wong ED, Zhu J, DeRisi JL: The transcriptome of the intraerythrocytic developmental cycle of Plasmodium falciparum. PLoS Biol 2003, 1(1):E5.
Label
Cy5
Label protocol
RNA extraction, cDNA synthesis and labeling as well as microarray hybridizations of the four samples representing time points 1, 2, 3, and 4 against a reference RNA pool were carried out as described in Bozdech Z, Llinas M, Pulliam BL, Wong ED, Zhu J, DeRisi JL: The transcriptome of the intraerythrocytic developmental cycle of Plasmodium falciparum. PLoS Biol 2003, 1(1):E5.
Synchronzied parasite cultures are splitted into proper number flasks (75ml culture/flask). Total number is double number of time points for one experiment. Half flasks are treated with one compound with the final concentration of IC50 or IC90, and half flasks are negative controls. After the treatment of different time points, parasites in each flask were harvested for total RNA isolation and microarray hybridizations.
Growth protocol
Laboratory strain 3D7 and Dd2, sorbitol synchronized. Parasites grow in a 2% suspension of purified human RBCs and RPMI 1640 media supplemented with 0.25% Albumax, 2 g/l sodium bicarbonate, 0.1 mM hypoxanthine, 25 mM HEPES (pH 7.4), and 50 μg/l gentamycin, at 37°C, 5% O2, and 6% CO2. The 3D7 parasites for microarray reference pool were mixed with 6 time-series samples (every 8hrs through a 48hr life cycle), harvested from a 6 liter biofermenter synchronized culture.
Extracted molecule
total RNA
Extraction protocol
RNA extraction, cDNA synthesis and labeling as well as microarray hybridizations of the four samples representing time points 1, 2, 3, and 4 against a reference RNA pool were carried out as described in Bozdech Z, Llinas M, Pulliam BL, Wong ED, Zhu J, DeRisi JL: The transcriptome of the intraerythrocytic developmental cycle of Plasmodium falciparum. PLoS Biol 2003, 1(1):E5.
Label
Cy3
Label protocol
RNA extraction, cDNA synthesis and labeling as well as microarray hybridizations of the four samples representing time points 1, 2, 3, and 4 against a reference RNA pool were carried out as described in Bozdech Z, Llinas M, Pulliam BL, Wong ED, Zhu J, DeRisi JL: The transcriptome of the intraerythrocytic developmental cycle of Plasmodium falciparum. PLoS Biol 2003, 1(1):E5.
Hybridization protocol
Microarray hybridizations were carried out as described in Bozdech Z, Llinas M, Pulliam BL, Wong ED, Zhu J, DeRisi JL: The transcriptome of the intraerythrocytic developmental cycle of Plasmodium falciparum. PLoS Biol 2003, 1(1):E5. In short, hybridizations were performed for 16 hours at 65°C using a Maui hybridization system (BioMicro Systems, Salt Lake City, Utah, USA).
Scan protocol
Scanning as described in Bozdech Z, Llinas M, Pulliam BL, Wong ED, Zhu J, DeRisi JL: The transcriptome of the intraerythrocytic developmental cycle of Plasmodium falciparum. PLoS Biol 2003, 1(1):E5. In short, scanning was performed with an Axon GenePix 4000B microarray scanner and associated GenePix Pro v6.0 software (Axon Instruments, Union City, California, USA).
Description
no additional information
Data processing
Data processing as described in Bozdech Z, Llinas M, Pulliam BL, Wong ED, Zhu J, DeRisi JL: The transcriptome of the intraerythrocytic developmental cycle of Plasmodium falciparum. PLoS Biol 2003, 1(1):E5. In short, Normalization and subsequent filtering for quality control were carried out using NOMAD database (http://derisilab.ucsf.edu). Spots were considered of good quality when they were unflagged and had a median intensity greater than the local background plus 2 times the standard deviation of the background for each dye channel. The values presented in the sample data represent log2-transformed ratios of the measurement in the sample channel (Cy5) divided by the corresponding measurement in the reference channel (Cy3, P. falciparum RNA reference pool).
