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Malaria resistance

ABSTRACT: Examination of molecular basis of malaria resistance. Spleens from malaria resistant AcB55 and AcB61strains compared with A/J mice. Animals were sacrificed and spleens were rapidly frozen in liquid nitrogen, and subsequently stored at 80oC. Tissues were mechanically homogenized with a polytron and total cellular RNA was extracted using a commercially available reagent (TMTRIzol; Invitrogen). The poly-(A)+ fraction of the RNA was isolated by chromatography with oligo d(T) cellulose beads. For cDNA labeling, either 25 mg of total RNA or 2.5 mg of poly-(A) RNA was converted into cDNA using reverse transcriptase (RT; Super Script II, Invitrogen) and alternatively Cy5 or Cy3-labeled dCTP (1mM, Perkin Elmer-Cetus/NEN, Boston) in a reaction mixture containing 1.5 mL oligo (dT) (100pmol/ mL), 3 mL dNTP-dCTP (6.67mM each), 1 mL dCTP (2mM), 4 mL DTT (100mM), 8 mL 5 X RT Buffer (Invitrogen, California). The reactions were carried out at 42¼C for 3 hrs, after which the RNA was degraded by the addition of 0.5 mL RNase A (1 mg/mL) and 1.5 mL RNaseH 5 units/mL). Labeled cDNA was separated from unincorporated nucleotides (Qiagen column) and further concentrated by evaporation under vacuum. The arrays were pre-hybridized for 1-2hrs with DIGEasy hybridization buffer (Roche) containing 10ug/ml denatured salmon sperm DNA, and 10ug/ml yeast tRNA. The Cy5 and Cy3 labelled cDNAs were combined and hybridized in the same medium and incubated with the arrays for 16-18hrs at 37Co. Finally, the arrays were washed 3 x 10 mins in 0.1 x SSC (20 X SSC is 3M sodium chloride, 0.3 M sodium citrate, pH 7.0), 0.1%SDS at 50Co, and 4 x 3min in 0.1 x SSC at room temperature, and dried by centrifugation. Fluorescent array images were collected for both Cy3 and Cy5 with a ScanArray fluorescent scanner and image intensity data were extracted and analyzed with QuantArray 3.0 analysis software. Quantification data was imported in GeneSpring (Silicon Genetics). The import format was modified to maintain information relevant to user-contributed flagging as well as QC parameter furnished by the array manufacturer. Dye swap and Intensity-based normalization (Lowess) were performed using the default settings. Keywords: other

ORGANISM(S): Mus musculus

PROVIDER: GSE709 | GEO | 2003/10/06

SECONDARY ACCESSION(S): PRJNA87677

REPOSITORIES: GEO

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Project description:This series repressents the data set from the paper "Expression microarray analysis and oligo array CGH of aquired gemcitabine resistance in mouse colon reveals selection for chromosomal aberrations". Biological material: One solid mouse tumor "Colon 26A" was routinely maintained by successive transplantation. A subset of mice with this tumor was treated at the maximum tolerable dosage of Gemcitabine and successively transplanted 5 times and similarly treated. The last passage was completly resistant and designated, "Colon 26G". Expression array: Total RNA was isolated from frozen mouse tumors using Tryzol. Single stranded cDNA was synthetized from 30 mg of total RNA by reverse transcription using aminoallyl labeled dUTP (Ambion). Labelling was performed according to the aminoallyl labeling protocol. Briefly, cDNA was incubated at room temperature for 1 h with fluorolink monofunctional cy5 or cy3 (Amersham) followed by 15 min 4 M hydroxylamine treatment. Uncoupled dyes were removed using Qiaquick PCR purification columns (Qiagen) and mixed with 12 mg poly(dA) (Amersham), 60 mg yeast tRNA (Sigma) and 24 mg Cot-1 DNA (Invitroen). The probe was dissolved in 127 ml hybridization mixture containing 46% formamide, 9.5% dextran sulfate, 2x SSC and 0.2% SDS. The probe was heated to 70°C for 10 min and annealed at 37°C for 1 h. Slides were hybridized for 14 h at 37°C over-night (HyArray 12™, Perkin Elmer). After hybridization the slides were washed in the HybArray 12™ with 50% formamide, 2x SSC, pH 7 at 35°C for 15 min, followed b PI buffer (0.1 M sodium phosphate, 0.1% Igepal Ca630, pH 8) at room temperature and 3 washes of 0.2x SSC, 0.1x SSC and 0.01x SSC at room temperature followed by centrifugation. Arrays were scanned using a laser scanner (ScanArray Express, Perkin Elmer) and analysed using ImaGene version 5.6. Cy5 cy3 ratios are calculating by taking the log2 of the signal mean of each spot minus the median background mean of all spots on the array. This is followed by a standard normalization for spot intensity and calculation of the ratios. Keywords: other

Project description:To identify mRNAs associated with human PUM2 protein, we used a modified Ribonucleoprotein-ImmunoPrecipitation Microarray (RIP-Chip) approach on HeLa S3 cancer cells that express PUM2. PUM ribonucleoprotein (RNP) complexes were captured from cell-free extracts with specific anti PUM2 antibody coupled to protein A sepharose beads, and then eluted with SDS-EDTA. To control for non-specifically enriched RNAs, the same procedure was performed with beads that were not coupled with immunoprecipitating antibodies (mock samples). Total RNA was isolated from cell extracts and immunopurified samples with the mirVanaTM PARISTM kit (Ambion). RNA was quantified with a NanoDrop device (Witeg AG). Poly-adenylated RNAs were amplified in the presence of aminoallyl-UTP with Amino Allyl MessageAmp II aRNA kit (Ambion). For this purpose, 500 ng total RNA from extracts and half (50-100 ng) of the immunopurified RNAs were used for amplification. 8 ug of the amplified RNAs (aaRNA) were fluorescently labeled with NHS-monoester Cy3 and Cy5 dyes (GE HealthSciences), except for mock RNA samples, where an aaRNA amount proportional to the yield obtained from corresponding PUM affinity isolates was used.For PUM2 RIPs, we performed four biological replicates but omitted the dye swaps due to the lower aaRNA obtained after amplification (~10 ug aaRNA from PUM2 RIPs, 9 ug aaRNA from mock RIPs). The Cy3- and Cy5-labeled aaRNA samples were mixed and hybridized to human cDNA microarrays. Detailed methods for microarray experiments are available at http://cmgm.stanford.edu/pbrown/protocols/index.html. cDNA microarrays were produced by the Stanford Functional Genomic Facility and contained 43,197 human probes representing 26,524 Unigene cluster IDs (12,466 ENSEMBL annotated genes) spotted on Corning Ultra GAPS slides. Spotted cDNAs were cross-linked with 65 mJ of UV irradiation on slides, which were then post-processed for 1 hour at 42°C in pre-hybridization solution (5x SSC, 0.1% SDS, 0.1 mg/ml BSA), washed twice in 400 ml of 0.1x SSC for 5 min, dunked in 400 ml ultrapure water for 30 sec, and dried by centrifugation at 550 rpm for 5 min. Slides were used the same day. Cy3- and Cy5-labeled aaRNA probes were mixed and applied to arrays in hybridization solution (3x SSC, 20 ug poly(A) RNA [Invitrogen], 20 ug yeast tRNA [Invitrogen], 20 ug Human Cot-1 DNA [Invitrogen], 20 mM HEPES [pH 7.0] and 0.3% SDS) for 18 h at 65°C. The arrays were then washed sequentially in 400 ml of 2x SSC with 0.1% SDS, 1x SSC, and 0.2x SSC. The first wash was performed for 5 min at 65°C, the subsequent washes were performed for 5 min at RT. The arrays were dried by centrifugation and immediately scanned with an AxonScanner 4200A (Molecular Devices). Data were collected using GENEPIX 5.1 (Molecular Devices). Arrays were normalized computationally by the Stanford Microarray Database (SMD). The data were filtered for signal over background of greater than 1.5 in the channel measuring aaRNA from extract, and only features that met these criteria in > 50% of the arrays were included for further analysis. Log2 median ratios were retrieved and exported into Microsoft Excel. To identify transcripts that were specifically enriched by association with PUM2, we performed two class Significance Analysis of Microarrays (SAM) on median centered arrays. A replicate experimental design type is where a series of replicates are performed to evaluate reproducibility or as a pilot study to determine the appropriate number of replicates for a subsequent experiments. Antibody used in IP: 50 ug rabbit anti-PUMILIO 2 (Bethyl Laboratories, #300-202A) Keywords: replicate_design

Project description:To identify mRNAs associated with human PUM1 protein, we used a modified Ribonucleoprotein-ImmunoPrecipitation Microarray (RIP-Chip) approach on HeLa S3 cancer cells that express PUM1. PUM ribonucleoprotein (RNP) complexes were captured from cell-free extracts with anti PUM1 specific antibody Bethyl Laboratories, #300-201A coupled to protein G (PUM1) sepharose beads, and then eluted with SDS-EDTA. To control for non-specifically enriched RNAs, the same procedure was performed with beads that were not coupled with immunoprecipitating antibody (mock samples). Total RNA was isolated from cell extracts and immunopurified samples with the mirVanaTM PARISTM kit (Ambion). RNA was quantified with a NanoDrop device (Witeg AG). Poly-adenylated RNAs were amplified in the presence of aminoallyl-UTP with Amino Allyl MessageAmp II aRNA kit (Ambion). For this purpose, 500 ng total RNA from extracts and half (50-100 ng) of the immunopurified RNAs were used for amplification. 8 ug of the amplified RNAs (aaRNA) were fluorescently labeled with NHS-monoester Cy3 and Cy5 dyes (GE HealthSciences), except for mock RNA samples, where an aaRNA amount proportional to the yield obtained from corresponding PUM affinity isolates was used. For PUM1 RIPs, we performed three biological replicates with technical (dye swap) replicates (total six arrays).(~40 ug aaRNA from PUM1 RIPs, ~9 ug aaRNA from mock RIPs). The Cy3- and Cy5-labeled aaRNA samples were mixed and hybridized to human cDNA microarrays.Detailed methods for microarray experiments are available at http://cmgm.stanford.edu/pbrown/protocols/index.html. cDNA microarrays were produced by the Stanford Functional Genomic Facility and contained 43,197 human probes representing 26,524 Unigene cluster IDs (12,466 ENSEMBL annotated genes) spotted on Corning Ultra GAPS slides. Spotted cDNAs were cross-linked with 65 mJ of UV irradiation on slides, which were then post-processed for 1 hour at 42°C in pre-hybridization solution (5x SSC, 0.1% SDS, 0.1 mg/ml BSA), washed twice in 400 ml of 0.1x SSC for 5 min, dunked in 400 ml ultrapure water for 30 sec, and dried by centrifugation at 550 rpm for 5 min. Slides were used the same day. Cy3- and Cy5-labeled aaRNA probes were mixed and applied to arrays in hybridization solution (3x SSC, 20 ug poly(A) RNA [Invitrogen], 20 ug yeast tRNA [Invitrogen], 20 ug Human Cot-1 DNA [Invitrogen], 20 mM HEPES [pH 7.0] and 0.3% SDS) for 18 h at 65°C. The arrays were then washed sequentially in 400 ml of 2x SSC with 0.1% SDS, 1x SSC, and 0.2x SSC. The first wash was performed for 5 min at 65°C, the subsequent washes were performed for 5 min at RT. The arrays were dried by centrifugation and immediately scanned with an AxonScanner 4200A (Molecular Devices). Data were collected using GENEPIX 5.1 (Molecular Devices). Arrays were normalized computationally by the Stanford Microarray Database (SMD). The data were filtered for signal over background of greater than 1.5 in the channel measuring aaRNA from extract, and only features that met these criteria in > 50% of the arrays were included for further analysis. Log2 median ratios were retrieved and exported into Microsoft Excel.To identify transcripts that were specifically enriched by association with PUM1, we performed two class Significance Analysis of Microarrays (SAM) on median centered arrays. A replicate experimental design type is where a series of replicates are performed to evaluate reproducibility or as a pilot study to determine the appropriate number of replicates for a subsequent experiments. Antibody used in IP: 20 ug goat anti-PUMILIO 1 Bethyl Laboratories, #300-201A Keywords: replicate_design

Project description:Escherichia coli strain MG1655 was grown to mid-log phase in defined anaerobic media. One sample was treated with 100 ÂµM CORM-3, the other sample was a control. The volume (175 ml), temperature (37oC) and stirring (200 rpm) were constant. After 15 min of exposure to CO-RM, samples were taken from treated and control cells, harvested into ice cold phenol ethanol (187 Âµl phenol, 3.56 ml ethanol) to stabilize RNA, and total RNA was purified using Qiagenâs RNeasy Mini kit as recommended by suppliers. RNA was quantified using a BioPhotometer (Eppendorf). Biological experiments were carried out four times, and a dye swap performed for each experiment, providing two technical repeats for each of the four biological repeats. Cells were grown in defined minimal medium (anaerobic). Final concentrations are: glycerol (54 mM), K2HPO4 (23 mM), KH2HPO4 (7.3 mM), NH4Cl (18.7 mM), CaCl2 (69 ÂµM), K2SO4 (15 mM), sodium fumarate (50 mM), Luria Broth (5%) and can amino acids (0.1%). Trace elements were added at 10 ml per l; final concentrations are: 134 ÂµM EDTA, 31 ÂµM FeCl3, 6.15 ÂµM ZnO, CuCl2 0.57 ÂµM, CoNo3 0.34 ÂµM, 1.6 ÂµM H3BO3, 1 ÂµM ammonium molybdenate and 1 ÂµM sodium selenite. MgCl2 was added at 1 ml per l. All chemicals were of AnalaR grade of purity or higher. E. coli strain MG1655 was grown in mini fermenter vessels in 175 ml of the above defined minimal media (anaerobic). The vessel was continually sparged with N2 to ensure anaerobiosis. CORM-3 was added at a final concentration of 100 ÂµM, chosen because it causes only a slight reduction in the growth rate. Cells were grown to mid-log (OD600 of 0.4) before addition of CO-RM. For RNA extraction, 30 ml was harvested into ice cold phenol ethanol (187 Âµl phenol, 3.56 ml ethanol) to stabilize RNA, and total RNA was purified using Qiagenâs RNeasy Mini kit as recommended by suppliers. RNA was quantified using a BioPhotometer (Eppendorf). Biological experiments were carried out four times, and a dye swap performed for each experiment, providing two technical repeats for each of the four biological repeats. Equal quantities of RNA from control and CO-RM-treated cells were labelled by using nucleotide analogues of dCTP containing either Cy3 or Cy5 fluorescent dyes (Perkin Elmer). For each microarray slide, one sample was labelled with Cy3-dCTP, while the other sample was labelled with Cy5-dCTP. RNA (approximately 9 micrograms) was annealed to 4.5 micrograms pd(N)6 random hexamers (Amersham Biosciences) by heating at 65 oC for 10 min, followed by 10 min at 22 oC and cooling on ice for 2 min. This was supplemented with 6 microlitres of 5Ã First-strand buffer (Invitrogen), 2.5 microlitres dNTP mixture (5 mM each dATP, dTTP, dGTP and 2 mM dCTP), 3 microlitres 0.1 M DTT, 2 microlitres 1 mM Cy3 or Cy5 (Perkin Elmer) and 1.5 microlitre Superscript III reverse transcriptase (200 U) (Invitrogen). This was incubated at 42 oC for 3 hours. The reaction was terminated by the addition of NaOH (7.5 microlitres of 1M) and HCl (7.5 microlitres of 1M) before the addition of TE buffer (300 microlitres). Labelled cDNA was purified using a Qiaquick PCR purification kit (Qiagen). The Cy3-dCTP-labelled sample was mixed with the Cy5-dCTP-labelled sample, vacuum dried and re-suspended in 120 microlitres salt-based hybridisation buffer (supplied with the microarray slides). This was heated at 95 oC for 3 min then cooled on ice for 3 min. The mixture was pipetted onto a microarray slide, sealed with a GeneFrame and coverslip in a hybridization chamber and incubated for 18 h at 42 Â°C. Following hybridization, microarray slides (minus GeneFrame and coverslip) were washed in a series of pre-warmed (37 Â°C) SSC buffers for 5 min each at 37oC: 2Ã SSC/0.1 % SDS, 1Ã SSC, 0.2Ã SSC and 0.01Ã SSC. Microarray slides were dried by centrifugation at 1500 Ã g for 2 min before scanning. Slides were scanned on an Affymetrix 428 scanner. The average signal intensity and local background correction were obtained using a commercially available software package from Biodiscovery, Inc (Imagene, version 4.0 and GeneSight, version 3.5). Spots automatically flagged as bad, negative or poor in the Imagene software were removed before the statistical analysis was carried out in GeneSight. The mean values from each channel were log2 transformed and normalised using the Lowess method to remove intensity-dependent effects in the log2(ratios) values. The Cy3/Cy5 fluorescent ratios were calculated from the normalized values. Biological experiments (i.e. a comparison of control and plus CO-RM cells) were carried out four times, and a dye swap performed for each experiment, providing two technical repeats for each of the four biological repeats. Data from the independent experiments were combined. Genes that were differentially expressed â¥ twofold and displayed and P value of < 0.05 (as determined by a t test) were defined as being statistically significantly differentially transcribed.

Project description:MicroPoly(A)Pure kits (Ambion) were used to prepare mRNA from P. salmonis-infected and control Atlantic salmon head kidney. Head kidney mRNA samples were prepared from pooled (n = 10) infected or control kidney. Approximately 200 ng of infected or control head kidney mRNA was used as template in aRNA synthesis reactions. Two infected head kidney samples were pooled to yield 8.9 ug of aRNA, and a single control head kidney sample contained 8.4 ug of aRNA. Infected and control head kidney aRNA samples were visualized on agarose gels to check quality and quantity (data not shown). Except for the amounts of aRNA and reverse transcription (RT) primer used, head kidney target labeling reactions and microarray hybridizations were identical. Head kidney target syntheses used 2 ug of aRNA and 2 ug of random primers (Roche). RT primers were mixed with aRNA samples, and nuclease-free H2O (Invitrogen) was added to make 10 ul total volume. Samples were incubated at 70 degrees C for 4 min, then placed on ice. RT reactions included 50 mM Tris HCl pH 8.3, 75 mM KCl, 3 mM MgCl2, 10 mM DTT, 0.5 mM each dATP, dCTP, and dGTP, 0.2 mM dTTP, 0.05 mM Cy3-dUTP or Cy5-dUTP (Amersham), 40U RNAguard ribonuclease inhibitor (Amersham), and 400U Superscript II Rnase H- reverse transcriptase (Invitrogen). RT reactions were incubated at 42 degrees C for 2.5 h, followed by addition of 0.025U RNase A (Sigma) and 1.5U RNase H (New England BioLabs) and incubation at 37 degrees C for 30 min. Labeled targets were cleaned using the Qiaquick PCR purification kit (Qiagen) and precipitated, with 300 mM sodium acetate, 1 ul of 20 ug/ul glycogen (Invitrogen), and 2.5 volumes of 100% ethanol, at -20 degrees C overnight. Each labeled target was recovered by centrifugation (14,000 rpm, 4 degrees C, 1 h), washed in 70% ethanol, air-dried, and resuspended in 60 ul of hybridization buffer: 50% deionized formamide, 5X SSC, 0.1% SDS, 5 ul of 5 ug/ul oligo dT (5’T18[Q-N]3’), 5 ul of 2 ug/ul BSA (Pierce), and 2 ul of 10 ug/ul sonicated human placental DNA (Sigma). Targets were incubated at 96 degrees C for 3 min, and then at 65 degrees C until applied to microarrays. Microarrays were prepared for hybridization by washing 2 X 5 min in 0.1% SDS, washing 5 X 1 min in MilliQ H2O, immersing 3 min in 95 degrees C MilliQ H2O, and drying by centrifugation (2000 rpm, 5 min, in 50 ml conical tube). Microarray hybridizations were run in the dark under HybriSlips hybridization covers (Grace Biolabs) in slide hybridization chambers (Corning) submerged in a 45 degrees C water bath for 16 h. Coverslips were floated off in 45 degrees C (1X SSC, 0.2% SDS), and arrays were washed 1 X 10 min in 45 degrees C (1X SSC, 0.2% SDS), 3 X 4 min in room temperature (0.1X SSC, 0.1% SDS), and 4 X 4 min in room temperature 0.1X SSC. Slides were dried by centrifugation as before. Fluorescent images of hybridized arrays were acquired immediately at 10 um resolution using ScanArray Express (PerkinElmer). The Cy3 and Cy5 cyanine fluors were excited at 543 nm and 633 nm respectively, and the same laser power (90%) and photomultiplier tube (PMT) settings were used for all slides in a study (PMT 75 Cy3, PMT 63 or 64 Cy5 for head kidney study). Fluorescent intensity data were extracted from TIF images using Imagene 5.5 software (Biodiscovery). Keywords: other

Project description:Subconfluent AG01518 fibroblasts were starved for 16 hours in the presence of 0.1% BSA, and then stimulated for 1, 4, 10 or 24 hours with PDGF-BB (10 ng/ml in starvation medium), or left untreated for the same periods of time. Total RNA was isolated using the RNeasy kit (Qiagen). The RNA quality was checked by formaldehyde agarose gel electrophoresis (RNeasy protocol, Qiagen). Total RNA (40 mg) from cells treated for a given period of time with PDGF or starvation medium alone were labelled in reverse transcription reactions (Superscript II kit, Invitrogen) with dCTP-Cy5 and dCTP-Cy3, respectively (Amersham). In every second replicate experiment the fluorescent deoxynucleotides were swapped. Purified cDNA probes labelled with Cy3 and Cy5 were mixed per pair, and hybridized to cDNA microarray chips (hver1.2.1) from the Sanger Institute/LICR/CRUK Consortium (see http://www.sanger.ac.uk/Projects/Microarrays/ for details and hybridization protocols). For each time point, we performed at least four independent hybridizations, and used at least two different batches of RNA. Chips were scanned in a Perkin Elmer/GSI Lumonics ScanArray 4000 scanner and spot intensities were measured using the QuantArray software (histogram method with background subtraction). Normalization and statistical analysis of the quadruplicate data sets were performed using GeneSpring 5.0 analysis software (Silicon Genetics). A Lowess non-linear normalization was applied and the median of the ratio distribution for each array was set to 1. Regulated spots were selected based on the average ratio values above 1.750 for up-regulated genes and below 0.571 for down-regulated genes. In addition, we considered only genes that were significantly regulated (t-test, p<0.05) based on replicate hybridizations (global error model, GeneSpring). For all features selected using this protocol, the signal was significantly above the background, indicating that the expression of these genes was detectable. Finally, Hver1.2.1 microarrays contain replicate spots (1 to 6) corresponding to the same gene. Genes represented by spots that were not regulated in a similar manner were discarded. We show the average ratio of one representative spot for each regulated gene, with standard error calculated from multiple hybridizations and with the annotation provided by the microarray facility (Hver1.2.1_NCBI33). Keywords: time-course

Project description:Escherichia coli strain MG1655 was grown to mid-log phase in defined aerobic media. One sample was treated with 30 ÂµM CORM-3, the other sample was a control. The volume (30 ml), temperature (37oC) and shaking (200 rpm) were constant. After 15 min of exposure to CO-RM, samples were taken from treated and control cells, harvested into ice cold phenol ethanol (187 Âµl phenol, 3.56 ml ethanol) to stabilize RNA, and total RNA was purified using Qiagenâs RNeasy Mini kit as recommended by suppliers. RNA was quantified using a BioPhotometer (Eppendorf). Biological experiments were carried out four times, and a dye swap performed for each experiment, providing two technical repeats for each of the four biological repeats. Cells were grown in defined minimal medium (aerobic). Final concentrations are: glycerol (54 mM), K2HPO4 (23 mM), KH2HPO4 (7.3 mM), NH4Cl (18.7 mM), CaCl2 (69 ÂµM), and K2SO4 (15 mM). Trace elements were added at 10 ml per l; final concentrations are: 134 ÂµM EDTA, 31 ÂµM FeCl3, 6.15 ÂµM ZnO, CuCl2 0.57 ÂµM, CoNo3 0.34 ÂµM, 1.6 ÂµM H3BO3, 1 ÂµM ammonium molybdenate and 1 ÂµM sodium selenite. MgCl2 was added at 1 ml per l. All chemicals were of AnalaR grade of purity or higher. E. coli strain MG1655 was grown in 250 ml side arm flasks in 30 ml of the above defined minimal media (aerobic). CORM-3 was added at a final concentration of 30 ÂµM, chosen because it causes only a slight reduction in the growth rate. Cells were grown to mid-log (Klett values of 30-40) before addition of CO-RM. The total 30 ml volume was harvested into ice cold phenol ethanol (187 Âµl phenol, 3.56 ml ethanol) to stabilize RNA, and total RNA was purified using Qiagenâs RNeasy Mini kit as recommended by suppliers. RNA was quantified using a BioPhotometer (Eppendorf). Biological experiments were carried out four times, and a dye swap performed for each experiment, providing two technical repeats for each of the four biological repeats. Equal quantities of RNA from control and CO-RM-treated cells were labelled by using nucleotide analogues of dCTP containing either Cy3 or Cy5 fluorescent dyes (Perkin Elmer). For each microarray slide, one sample was labelled with Cy3-dCTP, while the other sample was labelled with Cy5-dCTP. RNA (approximately 9 micrograms) was annealed to 4.5 micrograms pd(N)6 random hexamers (Amersham Biosciences) by heating at 65 oC for 10 min, followed by 10 min at 22 oC and cooling on ice for 2 min. This was supplemented with 6 microlitres of 5Ã First-strand buffer (Invitrogen), 2.5 microlitres dNTP mixture (5 mM each dATP, dTTP, dGTP and 2 mM dCTP), 3 microlitres 0.1 M DTT, 2 microlitres 1 mM Cy3 or Cy5 (Perkin Elmer) and 1.5 microlitre Superscript III reverse transcriptase (200 U) (Invitrogen). This was incubated at 42 oC for 3 hours. The reaction was terminated by the addition of NaOH (7.5 microlitres of 1M) and HCl (7.5 microlitres of 1M) before the addition of TE buffer (300 microlitres). Labelled cDNA was purified using a Qiaquick PCR purification kit (Qiagen). The Cy3-dCTP-labelled sample was mixed with the Cy5-dCTP-labelled sample, vacuum dried and re-suspended in 120 microlitres salt-based hybridisation buffer (supplied with the microarray slides). This was heated at 95 oC for 3 min then cooled on ice for 3 min. The mixture was pipetted onto a microarray slide, sealed with a GeneFrame and coverslip in a hybridization chamber and incubated for 18 h at 42 Â°C. Following hybridization, microarray slides (minus GeneFrame and coverslip) were washed in a series of pre-warmed (37 Â°C) SSC buffers for 5 min each at 37oC: 2Ã SSC/0.1 % SDS, 1Ã SSC, 0.2Ã SSC and 0.01Ã SSC. Microarray slides were dried by centrifugation at 1500 Ã g for 2 min before scanning. Slides were scanned on an Affymetrix 428 scanner. The average signal intensity and local background correction were obtained using a commercially available software package from Biodiscovery, Inc (Imagene, version 4.0 and GeneSight, version 3.5). Spots automatically flagged as bad, negative or poor in the Imagene software were removed before the statistical analysis was carried out in GeneSight. The mean values from each channel were log2 transformed and normalised using the Lowess method to remove intensity-dependent effects in the log2(ratios) values. The Cy3/Cy5 fluorescent ratios were calculated from the normalized values. Biological experiments (i.e. a comparison of control and plus CO-RM cells) were carried out four times, and a dye swap performed for each experiment, providing two technical repeats for each of the four biological repeats. Data from the independent experiments were combined. Genes that were differentially expressed â¥ twofold and displayed and P value of < 0.05 (as determined by a t test) were defined as being statistically significantly differentially transcribed.

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Malaria resistance

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