Investigation Title	Transcription profiling of genetically cerebral malaria-resistant (CM-R) and cerebral malaria-susceptible (CM-S) mouse strains
Comment[Submitted Name]	TAGC - M. musculus - Cerebral Malaria
Experimental Design	strain_or_line_design	pathogenicity_design	disease_state_design	transcription profiling by array
Experimental Design Term Source REF	mo	mo	mo	EFO
Comment[ArrayExpressReleaseDate]	2005-06-30
Comment[AEMIAMESCORE]	5
Comment[ArrayExpressAccession]	E-MEXP-370
Comment[MAGETAB TimeStamp_Version]	2011-06-28 20:56:01 Last Changed Rev: 14857
Experimental Factor Name	mouse strain
Experimental Factor Type	strain_or_line
Experimental Factor Term Source REF
Person Last Name	Coltel	Delahaye	Puthier	Rihet	Iraqi	Grau	Flori	Nguyen	Houlgatte
Person First Name	Nicolas	Nicolas Frédéric	Denis	Pascal	Fuad A.	Georges Emile	Laurence	Catherine	Rémi
Person Mid Initials	N	N		P	F	G	L	C	R
Person Email			puthier@tagc.univ-mrs.fr
Person Phone			(+33) 04 91 26 94 82
Person Fax			(+33) 04 91 26 94 30
Person Address			Parc Scientifique de Luminy Case 906
Person Affiliation	Laboratory of Immunopathology	IGPP (EA864)	TAGC	IGPP (EA864)	International Livestock Research Institute	Laboratory of Immunopathology	IGPP (EA864)	TAGC (ERM206)	TAGC (ERM206)
Person Roles			submitter
Person Roles Term Source REF
Quality Control Type	biological_replicate
Quality Control Term Source REF	The MGED Ontology
Replicate Type
Replicate Term Source REF
Normalization Type
Normalization Term Source REF
Date of Experiment
Public Release Date	2005-06-30
PubMed ID	16362897
Publication DOI	16362897
Publication Author List	Nicolas Frédéric Delahaye; Nicolas Coltel; Denis Puthier; Laurence Flori; Rémi Houlgatte; Fuad A. Iraqi; Catherine Nguyen; Georges Emile Grau; Pascal Rihet
Publication Title	Gene-Expression Profiling Discriminates between Cerebral Malaria (CM) Susceptible Mice and CM-Resistant Mice
Publication Status	journal_article
Publication Status Term Source REF
Experiment Description	Gene expression patterns were investigated in well-defined genetically cerebral malaria-resistant (CM-R) and cerebral malaria-susceptible (CM-S) mouse strains. cDNA microarrays were used to search for differentially expressed genes in mouse brain. Four mouse strains, known to differ in susceptibility to cerebral malaria upon Plasmodium berghei ANKA infection, were compared: BALB/c and DBA/2 mice are CM-R, while C57BL/6 and CBA/J mice are CM-S.
Protocol Name	P-MEXP-9262	P-MEXP-1058	P-MEXP-9392	P-MEXP-9393	P-MEXP-9329	P-MEXP-9264
Protocol Type	specified_biomaterial_action		labeling	hybridization	feature_extraction	bioassay_data_transformation
Protocol Description	Six to 8 weeks old BALB/c, DBA/2 (cerebral malaria resistant (CM-R) strains), C57BL/6 and CBA/J (cerebral malaria susceptible (CM-S) strains) female, were obtained from IFFA CREDO (Ch. River Lab, France) and kept in our facilities. Five from each strain were infected by i.p. injection of Plasmodium berghei ANKA. Parasitaemia was monitored daily on blood smear. The CM-S mice developed a neurological syndrome which occured 6 to 7 days after parasite inoculation with a cumulative mortality of 100%. The CM-R mice die during the 3rd or the 4th week of infection, with severe anaemia and hyperparasitaemia. Brains were taken from CM-R and CM-S mice when the CM-S mice develop cerebral malaria. Brains were completely removed and cut in two parts: one part was frozen in RNALater (Qiagen, TM) until RNA extraction using Trizol reagent (Gibco BRL), and the other one was embedded in Tissue Tek (Leica), snap frozen in liquid nitrogen and kept at -80°C until histopathological examination of cryosections.	0.2 ml of chloroform was added to each sample and after vigorous shaking for 15 seconds they were centrifuged at 12,000 × g for 15 minutes at 4°C. The aqueous phase was recovered and 0.5 ml isopropyl alcohol were added. Samples were incubated at room temperature for 10 minutes and centrifuged at 12,000 × g for 10 minutes at 4°C. Pellet was washed with 1 ml 75% ethanol and centrifuged at 7,500 × g for 5 minutes at 4°C. Air-dried pellet was then resuspended in DEPC (diethyl pyrocarbonate) treated sterile distilled water.	Two types of 33P-labelled probes were used: the vector probe which is an oligonucleotide sequence common to all spotted PCR products and which allows to determine precisely the amount of target DNA accessible to hybridization in each spot, and the complex probes made from 5 µg of retrotranscribed total RNA.<br> <br> For vector probe labelling, 1 ml of LBP9 oligo (5' ACTGGCCGTCGTTTTACA 3') was mixed with: 5 µl of 5x Forward Reaction Buffer, 3 µl of g33P ATP, 1 µl of T4 polynucleotide kinase (Invitrogen) and 15 µl of water. After 10 minutes at 37°C, enzyme was heat-inactivated (10 min, 65°C). Unincorporated nucleotides were removed by purification on a G25 column.<br> <br> For complex probe labelling, 5 µg of total RNA was mixed with 8 µg oligodT(25) and 0.3 ng cytochrome c554 mRNA. Samples were heated at 70°C to remove secondary structure and progressively cooled to 42°C to ensure annealing of oligo(dT) with the poly(A) tail. Then, 17 µl of labeling mix (1 µl RNasin, 6 µl 5x first strand buffer, 2 µl 0.1 M DTT, 0.6 µl of 20 mM (each) dATP dGTP dTTP, 0.6 µl of 120 µM dCTP, 3 µl of alpha-33P-dCTP, 1 µl SuperScript II reverse transcriptase, 2.8 µl water) were added and reverse transcription was performed at 42°C for one hour. To improve reverse transcription, 1 µl SuperScript II reverse transcriptase was subsequently added and reaction was carried on for one hour. RNA was removed by treatment at 68°C for 30 min with 1 µl 1% SDS, 1 µl 0.5 M EDTA and 3 µl 3 M NaOH. After equilibration (15 min at room temperature) samples were neutralized using 10 µl 1 M Tris-HCl and 3 µl 2 N HCl. Next, 2 µg of polyA(80) and 2 µg of mouse COT-1 (Invitrogen) were added and samples were heated at 100°C for 5 min. Volume was then adjusted to 500 µl with hybridization buffer (5x SSC, 5x Denhardt's, 0.5% SDS) and samples were heated for 2 hr 30 at 68°C.	Each microarray was hybridized first with the vector probe, then after stripping, with a complex probe made from total RNA.<br> <br> For the vector probe hybridization, filters were pre-hybridized for 4 hours at 42°C in hybridization buffer containing 100 µg/ml denaturated (10 min at 100°C) herring sperm DNA. 50.000 cpm of the labeled oligonucleotide were added and hybridization was conducted for 10 hours at 42°C. Filters were then washed for 10 min at room temperature and 5 min at 42°C in 2X SSC/0.1% SDS solution. Filters were rinsed in 2X SSC buffer and air-dried before exposure.<br> <br> The vector probe was then stripped to allow the complex probe hybridization. Filters were washed at 68°C in 2X SSC/0.1% SDS solution for 3 hours. Filters were rinsed in 2X SSC buffer and air-dried before a control exposure.<br> <br> For the complex probe hybridization, filters were pre-hybridized for 6 hours at 68°C in hybridization buffer containing 100 µg/ml denaturated (10 min at 100°C) herring sperm DNA. Buffer was then removed and filters were hybridized with the labeled samples for 48 hours at 68°C. Filters were then washed at 68°C in 0.1X SSC/0.2% SDS solution for 3 hours. Finally, membranes were rinsed in 2X SSC and air-dried before exposure.	After hybridizations and washes, arrays were exposed for 48 hours to phosphor-imaging plates (FUJIFILM), which were then scanned with a FUJI BAS-5000 machine (25 µm resolution, dynamic range: L5/S10000). Hybridization signals were quantified using ArrayGauge software (Fuji Ltd, Tokyo, Japan).	After image acquisition, all images were carefully inspected, and spots with overestimated intensities due to neighborhood effects were manually excluded (flagged as "excluded" in the Overshining Status column). The data from 1 sample (CBA/J mouse) indicated a lower hybridization quality, so the sample was omitted from subsequent analyses.<br> The data were filtered such that only spots with intensities that were two times greater than the median background (median value of empty controls) in either microarray were used in the analysis. The signal intensities were then corrected to take into account the amount of spotted DNA and the variability of experimental conditions. Briefly, complex probe intensity of each spot ("C") was adjusted ("C/V") for the amount of target DNA accessible to hybridization as measured by using vector hybridization ("V"). When "V" intensity of a spot was too weak on a microarray, the corresponding cDNA clone was not considered for this experiment (missing data). Of the 5053 spotted clones, we selected the clones that had detectable expression levels in at least 80% of the experiments (n=2145). Data were log2-transformed and centered relative to the median for each gene and each array using the Cluster software (Eisen, 1998). Unsupervised hierarchical clustering (average linkage clustering using Pearson's uncentered correlation as similarity metric) was applied to the dataset and results were visualized with the Treeview software (Eisen, 1998).
Protocol Parameters		Amplification;Extracted product;	Amount of nucleic acid labeled;Label used;Amplification;	temperature;Chamber type;Quantity of label target used;Volume;time;
Protocol Hardware					FUJIFILM BAS-5000 scanning hardware
Protocol Software					ArrayGauge
Protocol Contact
Protocol Term Source REF	mo
SDRF File	E-MEXP-370.sdrf.txt
Term Source Name	mo		ncbitax	atcc	nci_thesaurus	NCI_thesaurus	ArrayExpress	The MGED Ontology	mo	EFO
Term Source File	http://mged.sourceforge.net/ontologies/MGEDontology.php		http://www.ncbi.nlm.nih.gov/Taxonomy/taxonomyhome.html	http://www.atcc.org/	http://nciterms.nci.nih.gov/NCIBrowser/Dictionary.do	ncithesaurus.obo.alt	http://www.ebi.ac.uk/arrayexpress	http://mged.sourceforge.net/ontologies/MGEDontology.php	http://mged.sourceforge.net/ontologies/MGEDontology.php	http://www.ebi.ac.uk/efo/
Term Source Version