Investigation Title Transcriptional analysis of wild-type Leishmania species between both stages using full-genome DNA microarrays Comment[Submitted Name] Transcriptional analysis of wild-type Leishmania species between both stages using full-genome DNA microarrays Experimental Design transcription profiling by array Experimental Design Term Source REF EFO Comment[SecondaryAccession] Comment[SecondaryAccession] GSE10407 Comment[ArrayExpressReleaseDate] 2008-05-28 Comment[AEMIAMESCORE] 4 Comment[Publication DOI] 10.1186/1471-2164-9-255 Comment[ArrayExpressAccession] E-GEOD-10407 Comment[MAGETAB TimeStamp_Version] 2010-10-22 19:33:50 Last Changed Rev: 14857 Experimental Factor Name ORGANISM Experimental Factor Type Organism Experimental Factor Term Source REF Person Last Name Ubeda Corbeil Boisvert Messier Papadopoulou Rochette Papadopoulou Rigault Smith Ouellette Raymond Person First Name J J S N B A Barbara P M M F Person Mid Initials M Person Email barbara.papadopoulou@crchul.ulaval.ca Person Phone 001-418-654-2705 Person Fax 001-418-654-2715 Person Address Centre de Recherche du CHUL, 2705 boulevard Laurier, Quebec, Quebec, Canada Person Affiliation Centre de Recherche du CHUL Person Roles submitter Person Roles Term Source REF The MGED Ontology Quality Control Type Quality Control Term Source REF Replicate Type Replicate Term Source REF Normalization Type Normalization Term Source REF Date of Experiment Public Release Date 2008-05-28 PubMed ID 18510761 Publication DOI 18510761 Publication Author List Rochette A, Raymond F, Ubeda JM, Smith M, Messier N, Boisvert S, Rigault P, Corbeil J, Ouellette M, Papadopoulou B Publication Title Genome-wide gene expression profiling analysis of Leishmania major and Leishmania infantum developmental stages reveals substantial differences between the two species. Publication Status journal_article Publication Status Term Source REF The MGED Ontology Experiment Description Transcriptional analyses of L. infantum promastigote compared to L. infantum intracellular amastigote, and L. major promastigote compared to L. major intracellular amastigote The full-genome DNA microarray includes one 70mer-oligonucleotide probe for each gene of L. infantum and for each gene of L.major LV39 Keywords: stage-specific comparison Leishmania infantum: Two-condition experiment, promastigote stage vs amastigote stage. Six biological replicates for each stage, independently grown and harvested. One replicate per array Leishmania major: Two-condition experiment, promastigote stage vs amastigote stage. Four biological replicates for each stage, independently grown and harvested. One replicate per array Protocol Name P-GSE10407-16 P-GSE10407-5 P-GSE10407-17 P-GSE10407-6 P-GSE10407-7 P-GSE10407-9 P-GSE10407-10 P-GSE10407-8 P-GSE10407-18 P-GSE10407-19 P-GSE10407-11 P-GSE10407-21 P-GSE10407-12 P-GSE10407-14 P-GSE10407-13 P-GSE10407-20 P-GSE10407-3 P-GSE10407-2 P-GSE10407-1 P-GSE10407-4 Protocol Type grow grow nucleic_acid_extraction nucleic_acid_extraction labeling hybridization hybridization hybridization hybridization image_aquisition image_aquisition feature_extraction feature_extraction feature_extraction feature_extraction feature_extraction bioassay_data_transformation bioassay_data_transformation bioassay_data_transformation bioassay_data_transformation Protocol Description Promastigote cells were grown in SDM-79 medium supplemented with 10% heat-inactivated fetal bovine serum and 5 µg/ml hemin at 25 °C. Amastigote cells were isolated from mice infected. Promastigote cells were grown in SDM-79 medium supplemented with 10% heat-inactivated fetal bovine serum and 5 µg/ml hemin at 25 °C. Amastigote cells were grown in THP-1 macrophage in RMPI 1640 medium supplemented with 10% heat-inactivated fetal bovine. Total RNA was isolated from 108 Leishmania cells during the mid-log phase (promastigote) or after 6 weeks of mice infection (amastigote) using RNeasy Plus Mini Kit (QIAGEN) as described by the manufacturer. The RNA preparation was treated with TURBO DNase (Ambion) to avoid any genomic contamination Total RNA was isolated from 108 Leishmania cells during the mid-log phase (promastigote) or after 4 days macrophage infection (amastigote) using RNeasy Plus Mini Kit (QIAGEN) as described by the manufacturer. The RNA preparation was treated with TURBO DNase (Ambion) to avoid any genomic contamination 10 µg of RNA were converted to aminoallyl-dUTP incorporated cDNA using random hexamers (GEHealthcare) and the SuperScript III RNase H- Reverse Transcriptase (Invitrogen). Probes were thereafter coupled to the fluorescent dye Alexa Fluor555 or Alexa Fluor647 (Invitrogen) following manufacturer recommendations. Fluorescent probes were then purified with MinElute Spin Columns (QIAGEN) and quantified spectrophotometrically. Slides were prehybridized in 5X Denhardt, 30% formamide, 6X SSPE, 0,5% SDS, 100µg/ml salmon sperm DNA. Then, slides were washed . For hybridization, labelled cDNA probes were dried and resuspended in 2,5X Denhardt, 30% formamide, 6X SSPE, 0,5% SDS, 100µg/ml salmon sperm DNA, 750µg/ml Yeast tRNA, and then mixed. Mixed probes were applied on the array under a lifterslip. Hybridization was performed for 16h. Prehybridization and hybridization were performed at 42°C. For hybridization, labelled cDNA probes were dried and resuspended in 2,5X Denhardt, 30% formamide, 6X SSPE, 0,5% SDS, 100µg/ml salmon sperm DNA, 750µg/ml Yeast tRNA. then mixed. Mixed probes were applied on the array under a lifterslip. Hybridization was performed for 16h. The Perkin Elmer ScanArray 4000XL Scanner was used for image acquisition. GenePix Pro 6.0 image analysis software (Axon Instruments) was used to quantify the fluorescence signal intensities of the array features The Perkin Elmer ScanArray 4000XL Scanner was used for image acquisition. GenePix Pro 6.0 image analysis software (Axon Instruments) was used to quantify the fluorescence signal intensities of the array features. Multiple testing corrections were done using the FDR method with a threshold p-value of 0.05. Only genes statistically significant with an absolute log ratio greater than 0.58 (log2 1.5) were considered as differentially expressed Raw data from GenePix were imported in R 2.2.1 for normalization, and statistical analyses were performed using the LIMMA (version 2.7.3) package. Multiple testing corrections were done using the FDR method with a threshold p-value of 0.05. Only genes statistically significant with an absolute log ratio greater than 0.75 (log2 1.7) were considered as differentially expressed Intra-array normalization was carried out using the ‘print-tip loess’ statistical method and inter-array normalization was done by using the ‘quantiles of A’ method for each array (Yang, 2003). Raw data from GenePix were imported in R 2.2.1 for normalization and statistical analyses were performed using the LIMMA (version 2.7.3) package. ID_REF =
VALUE = normalized log2 ratio (Alexa555/Alexa647) ID_REF =
VALUE = normalized log2 ratio (Alexa555/Alexa647)
INV_VALUE = normalized log2 ratio (Alexa647/Alexa555) ID_REF =
VALUE = normalized log2 ratio (Alexa647/Alexa555) ID_REF =
VALUE = normalized log2 ratio (Alexa647/Alexa555)
INV_VALUE = normalized log2 ratio (Alexa555/Alexa647) Protocol Parameters Protocol Hardware Protocol Software Protocol Contact Protocol Term Source REF The MGED Ontology The MGED Ontology The MGED Ontology The MGED Ontology The MGED Ontology The MGED Ontology The MGED Ontology The MGED Ontology The MGED Ontology The MGED Ontology The MGED Ontology The MGED Ontology The MGED Ontology The MGED Ontology The MGED Ontology The MGED Ontology The MGED Ontology The MGED Ontology SDRF File E-GEOD-10407.sdrf.txt Term Source Name ncbitax The MGED Ontology ArrayExpress EFO The MGED Ontology Term Source File http://www.ncbi.nlm.nih.gov/Taxonomy/taxonomyhome.html http://mged.sourceforge.net/ontologies/MGEDontology.php http://www.ebi.ac.uk/arrayexpress http://www.ebi.ac.uk/efo/ http://mged.sourceforge.net/ontologies/MGEDontology.php Term Source Version