In the three weeks prior to RNA collection, sorbitol lysis was carried out twice at 12 hour intervals in every asexual cycle. For the time course, schizont stage cultures were examined hourly until the first ring was seen, then sorbitol lysis was carried out 12 hours later, giving ring stage parasites within an 12-hour time window. Samples were collected for RNA immediately after sorbitol lysis (time point 1), and then 8-hourly, resulting in 6 time points covering the entire intra-erythrocytic life cycle.
Growth protocol
R29 was cultured at 2% haematocrit with group A erythrocytes. The culture medium used was RPMI 1640 medium supplemented with 25 mM Hepes, 2 mM glutamine, 25mM glucose, 25 μg/ml gentamicin, adjusted with 1M NaOH to pH 7.2 to 7.4 with 10% pooled normal human serum.
Extracted molecule
total RNA
Extraction protocol
The tube with TRIzol solution was thawed on ice. Two volumes chloroform (Sigma) were added per volume packed cells. After mixing, the tube was incubated on ice for 5min then centrifuged at 3600g for 40min at 4°C without brakes. The supernatant (aqueous layer) was carefully transferred into a fresh tube without disturbing the pellet. The same volume of ice-cold isopropanol (Sigma) was added and the tube was incubated at 4°C overnight. The next day, the tube was centrifuged at 3600g for 60 min at 4°C. The supernatant was discarded and the pellet was resuspended and washed with ice-cold 70% ethanol (Sigma). After another centrifugation at 3600g and 4°C for 10min, the supernatant was completely but carefully removed using a fine Pasteur pipette. The tube was left upside down to air dry for 15 to 60min until no liquid was visible. The dried pellet was resuspended with 25μl of warm DEPC-H2O then placed on ice. RNA concentration was measured using a spectrophotometer. For RNA, ten times the culture packed cell volume of room temperature TRIzol reagent (Invitrogen 15596-026) was added, and after thorough mixing, stored at -80°C. 12μg of RNA from the R29 non-rosetting parasites at each of the 6 time points was combined together to form the reference pool. The pool and 12μg of each individual time point sample from both rosetting and non-rosetting parasites were then used for first-strand cDNA synthesis using an amino-allyl dye coupling protocol
Label
Cy5
Label protocol
Each aminoallyl-cDNA sample was coupled to Cy5 (red dye) while Cy3 (green dye) was added to the pool. Cy5-labelled time point samples were mixed with the same amount of Cy3-labelled pool sample.
strain: IT/R29 sample type: rosetting developmental stage: Pool of the 6 timepoints
Treatment protocol
In the three weeks prior to RNA collection, sorbitol lysis was carried out twice at 12 hour intervals in every asexual cycle. For the time course, schizont stage cultures were examined hourly until the first ring was seen, then sorbitol lysis was carried out 12 hours later, giving ring stage parasites within an 12-hour time window. Samples were collected for RNA immediately after sorbitol lysis (time point 1), and then 8-hourly, resulting in 6 time points covering the entire intra-erythrocytic life cycle.
Growth protocol
R29 was cultured at 2% haematocrit with group A erythrocytes. The culture medium used was RPMI 1640 medium supplemented with 25 mM Hepes, 2 mM glutamine, 25mM glucose, 25 μg/ml gentamicin, adjusted with 1M NaOH to pH 7.2 to 7.4 with 10% pooled normal human serum.
Extracted molecule
total RNA
Extraction protocol
The tube with TRIzol solution was thawed on ice. Two volumes chloroform (Sigma) were added per volume packed cells. After mixing, the tube was incubated on ice for 5min then centrifuged at 3600g for 40min at 4°C without brakes. The supernatant (aqueous layer) was carefully transferred into a fresh tube without disturbing the pellet. The same volume of ice-cold isopropanol (Sigma) was added and the tube was incubated at 4°C overnight. The next day, the tube was centrifuged at 3600g for 60 min at 4°C. The supernatant was discarded and the pellet was resuspended and washed with ice-cold 70% ethanol (Sigma). After another centrifugation at 3600g and 4°C for 10min, the supernatant was completely but carefully removed using a fine Pasteur pipette. The tube was left upside down to air dry for 15 to 60min until no liquid was visible. The dried pellet was resuspended with 25μl of warm DEPC-H2O then placed on ice. RNA concentration was measured using a spectrophotometer. For RNA, ten times the culture packed cell volume of room temperature TRIzol reagent (Invitrogen 15596-026) was added, and after thorough mixing, stored at -80°C. 12μg of RNA from the R29 non-rosetting parasites at each of the 6 time points was combined together to form the reference pool. The pool and 12μg of each individual time point sample from both rosetting and non-rosetting parasites were then used for first-strand cDNA synthesis using an amino-allyl dye coupling protocol
Label
Cy3
Label protocol
Each aminoallyl-cDNA sample was coupled to Cy5 (red dye) while Cy3 (green dye) was added to the pool. Cy5-labelled time point samples were mixed with the same amount of Cy3-labelled pool sample.
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)
Data processing
All arrays were visually inspected using GenePix and any bad quality spots (signal below background or dust on the chip) were flagged out. After gridding, the data were loaded onto the Acuity 4.0 software. Each array was then normalized with Lowess (locally weighted least squares regression). A dataset with all time points was created using the following parameters: only unflagged spots and spots with median intensities (green or red) greater than the local background plus 2 times the standard deviation of the background were used.
