A whole-tissue RNA-seq toolkit for organism-wide studies of gene expression with PME-seq
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE138103 | GEO | 2019/10/01
REPOSITORIES: GEO
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Project description:Complex organisms are composed of organs and tissues which evolved to their present state as they function together to improve an organism's overall reproductive fitness. Studies of individual organs help us understand their basic functions but this reductionist approach misses the larger context of the whole organism. This problem can be circumvented if all the organs in an organism were comprehensively studied by the same methodology and analyzed together. Using honey bees (Apis mellifera L.) as a model system,we report here the first whole proteome of a complex organism,measuring 29 different tissue types among the three honey bee castes: queen,drone,and worker. The data reveal that,e.g.,workers have a heightened capacity to deal with environmental toxins and queens have a far more robust immune system than their nestmates. Most intriguingly,our analysis reveals the path by which organs of complex organisms probably evolved.Raw data were processed by MaxQuant (v1.2.0.13) using default parameters, plus lysine and N-terminal dimethylation for three isotopologues for relative quantitation. Data were searched against the Apis Official Gene Set version 2 with common contaminants, containing a total of 22037 sequences. Normalized intensity ratios were used for relative quantitation.
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2020-10-20 | GSE151754 | GEO
Project description:Array Design; Mouse Compugen 22K Spotted Oligo Author; Sean Grimmond Common Reference (Yes/No, ID); Yes Mouse universal Reference (Stratagene) Concentration; 100 Data processing/normalization; Lowess Normalisation GeneSpring) Developmental Stage; embryonic Diseased/Normal; normal Dye Swap; red/green Experiment Type; time course Genetic Characteristic; Wild type Image Analysis Software; Imagene5.5 (Biodiscovery) Labeling Protocol; Amino Allyl Message Amp- Ambion Organ/Organism part; kidney Organism; Mus Musculus RNA type; aRNA Message Amp-ambion Tissue Type; Pool of whole organs Organization; IMB, Uni of QLD, Brisbane Australia Sample Source; primary tissue Sampling Method; Dissection of whole organ Sex; Pool of both sexes Strain/Cell-line; CD1 Series_overall_design: To account for dye swap, the signal channel and control channel measurements for each sample were reversed. A Lowess curve was fit to the log-intensity versus log-ratio plot. 20.0% of the data was used to calculate the Lowess fit at each point. This curve was used to adjust the control value for each measurement. If the control channel was lower than 10 then 10 was used instead. Specific samples were normalized to one another: sample(s) 1-38 were normalized against the median of the control sample(s) 1-38. Each measurement for each gene in those specific samples was divided by the median of that gene's measurements in the corresponding control samples. Keywords: time-course
2005-07-20 | GSE2282 | GEO
Project description:Array Design; Mouse Compugen 22K Spotted Oligo Author; Sean Grimmond Common Reference (Yes/No, ID); Yes Mouse universal Reference (Stratagene) Concentration; 100 Data processing/normalization; Lowess Normalisation GeneSpring) Developmental Stage; embryonic Diseased/Normal; normal Dye Swap; red/green Experiment Type; time course Genetic Characteristic; Wild type Image Analysis Software; Imagene5.5 (Biodiscovery) Labeling Protocol; Amino Allyl Message Amp- Ambion Organ/Organism part; kidney Organism; Mus Musculus RNA type; aRNA Message Amp-ambion Tissue Type; Pool of whole organs Organization; IMB, Uni of QLD, Brisbane Australia Sample Source; primary tissue Sampling Method; Dissection of whole organ Sex; Pool of both sexes Strain/Cell-line; CD1 Series_overall_design: To account for dye swap, the signal channel and control channel measurements for each sample were reversed. A Lowess curve was fit to the log-intensity versus log-ratio plot. 20.0% of the data was used to calculate the Lowess fit at each point. This curve was used to adjust the control value for each measurement. If the control channel was lower than 10 then 10 was used instead. Specific samples were normalized to one another: sample(s) 1-38 were normalized against the median of the control sample(s) 1-38. Each measurement for each gene in those specific samples was divided by the median of that gene's measurements in the corresponding control samples. Keywords: time-course
2010-06-09 | E-GEOD-2282 | biostudies-arrayexpress
Project description:Experiment Design 1) Experiment Type normal vs diseased comparison, time course 2) Experimental Factor response to viral infection - SHIV89.6P 3) Number of hybridizations 60 4) Common Reference No 5) Quality Control Steps 4 technical replicate hybridizations per animal (3 replicate hybridizations for C89115M, C95067F) dual spotting of gene per array multiple spotting of select genes per array 2 biological replicates (2 different animals) per time point dye swaps 6) Experiment Description 4 cynomolgus monkeys (Macaca fascicularis) were infected with TCID50 of SHIV89.6P. Whole blood RNA was collected at 0, 1, 2, 4 and 5 weeks post-infection. Post-infection samples were competitively hybridized with pre-infected samples for each individual animal. Unpublished Data 7) Qualifier, value, source 2.2. Samples: samples used, extract preparation and labeling 2.2.1. Biosource properties 1) Organism (NCBI taxonomy) Macaca fascicularis 2) Contact details for sample Dr Erling W Rud Animal Resources Division, Health Canada. Sir Frederick Banting Research Centre Building 22, Room C308, postal locator 2203E Tunneys' Pasture Ottawa,Ontario, Canada K1A 0L2 Tel:(613) 957-8049 FAX:(613) 941-6625 erling_rud@hc-sc.gc.ca 3) Cell Type not applicable 4) Sex C88015F - female C89115M - male C94071M - male C95067F - female 5) Age C88015F - 13 years C89115M - 12 years C94071M - 7 years C95067F - 6 years 6) Developmental Stage adult 7) Organism Part peripheral whole blood 8) Strain or line not applicable 9) Genetic variation not applicable 10) Individual number C88015F, C89115M, C94071M & C95067F 11) Individual genetic characteristics not applicable 12) Disease state normal, AIDS 13) Targeted cell type blood 14) Cell line not applicable 2.2.2. Biomaterial manipulation 1) Growth conditions not applicable 2) In vivo treatments description - 4 cynomolgus monkeys (Macaca fascicularis) were infected intravenously with TCID50 of SHIV89.6P. 10 ml of peripheral blood was drawn at -2, 0, 1, 2, 4 and 5 weeks post-infection. 3) In vitro treatment not applicable 4) Treatment type infection 5) Compound not applicable 6) Separation technique not applicable 2.2.3. Hybridization extract preparation 1) Extraction method PAXgene peripheral blood RNA preparation 10 ml of peripheral blood was drawn from each animal directly into 4 PAXgene Blood RNA Tubes (QIAGEN #762125) according to manufacturer's protocol (http://www.qiagen.com/literature/Handbooks/PDF/DNA_RNA_isolation_clinical/INT/PAXgene_Blood_RNA/PaxgeneBloodRNAtube.pdf) Total RNA was purified using PAXgene Blood RNA kit (QIAGEN # 762134) according to manufacturer's protocol http://www.qiagen.com/literature/Handbooks/PDF/DNA_RNA_isolation_clinical/INT/PAXgene_Blood_RNA/1017455PAXgeneBloodRNA_prot.pdf) 2) Nucleic acid type total RNA 3) Amplification Method Total RNA was amplified using the Ambion MessageAmp aRNA kit (#1750), an Eberwine based method, with slight modifications to the manufacturer's protocol (http://www.transnet.ca/Amplification%20Protocol.htm). 2.2.3. Sample labeling 1) Amount of nucleic acid labeled 6 micrograms of aRNA was labeled with Cy3/Cy5 for hybridization 2) Label used Cy5 & Cy3 3) Label incorporation method aRNA was labelled by reverse transcription using the standard protocol of the Ontario Cancer Institute with slight modifications (http://www.transnet.ca/Hybridization%20Protocol_2.htm) 2.2.5. Spiking controls 1) Spiking control feature OCI Human 19k3-part1 OCI Human 19k3-part2 2) Spike type and qualifier not applicable 3) Qualifier, value, source not applicable 2.3. Hybridization procedure and parameters 2) Hybridization Protocol Protocol - (http://www.transnet.ca/Hybridization%20Protocol_2.htm) Solution Hybridization buffer: DIG solution (Roche cat.#1603558) supplemented with 0.45 ug/ul yeast tRNA, 0.45 ug/ul salmon sperm DNA Blocking agent not applicable Slide blocking not applicable Probe blocking: 0.45 ug/ul yeast tRNA, 0.45 ug/ul salmon sperm DNA during hybridization Wash procedure 3x15 min with 1xSSC, 0.1% SDS at 50 degrees Celsius, 3 fast washes in 1xSSC at 50 degrees Celsius Quantity of labelled target used all material generated from 6 ug amplified RNA per sample Time 18 hr (overnight) hybridization Concentration 0.2 ug/ul of aRNA per hybridization Volume 60 ul Temperature 30 degrees Celsius Description of the hybridization chamber hybridizations were incubated in a microscope slide box stored within a tissue culture incubator Qualifier, value, source not applicable 2.4. Measurements 2.4.1. Raw data Scanning protocol Scanning hardware: Scanarray Express Microarray Scanner, Packard Biosciences Scanning software: Scanarray Express Microarray Analysis System v1.1 Build #9 Scan Parameters Laser power: see individual image files in section 2.4.1 Spatial resolution:10 microns PMT gain: 60% 2.4.2. Image analysis and quantitation Image analysis software: Quantarray v.3.0, Packard BioSciences Availability: No longer commercially available Quantitation Parameters: Array Pattern: Spot rows: 25 Spot columns: 24 Spot spacing (vertical): 170 microns Spot spacing (horizontal): 170 microns Interstitial spacing: Off Array rows: 8 Array columns: 4 Array spacing (vertical): 4500 microns Array spacing (horizontal): 4500 microns Auto-adjustable Grid: Grid elasticity: 50/100 Spot elasticity: 50/100 Quantitation: Method: Fixed Circle Crosstalk correction: No Normalized to brightest: No Background subtraction: No Gene database file: for OCI Human 19k3-part1- H19k3a.qa (attached) for OCI Human 19k3-part2- H19k3b.qa (attached) Fixed circle quantitation parameters: Spot diameter: 60 microns Inner background diameter: 180 microns Outer background diameter: 200 microns Signal low percentile: 45 Signal high percentile: 95 Background low percentile: 5 Background high percentile: 55 Quantification output: Mean Intensity Quality Criteria Confidence calculation: Minimum Tolerance and weight: None selected Replicates: Combine replicates: no 2.4.3. Normalized and summarized data Data processing protocol Background subtraction and normalization Background subtraction and normalization was performed using the Quantarray Normalization Excel macro on individual quantitation output files. Each channel was background subtracted using the equation: ch Bkg. Subtr. Intensity = ch intensity - ch Bkg. Normalization strategy: total array Normalization algorithm: log ratio median centering Median log ratio = median(log2(ch1 Bkg. Subtr. Intensity) - log2(ch2 Bkg. Subtr. Intensity)) Normalization factor = 2^ median log ratio Normalized ch2 intensity = ch2 intensity*Normalization factor Statistical Analysis The background subtracted normalized intensities were used for statistical analysis. All spot intensities for within-chip, repeat hybridization, fluor-flip hybridization and biological replicates for each time point post-infection were compiled and tested for significance testing using the software package ArrayStat v.1.0, Imaging Research. ArrayStat parameters: Model selection: Manual Model selected: Proportional model with offsets Outlier detection: yes Threshold detection: Manual Threshold detected: p<0.01 Minimum No. of replicates: 12 (for 1 & 5 wks PI), 11 (for 2 & 4 wks PI) Conditions: Dependent Random Error Estimation Method: Small Sample Test: t-test Normalization: By median Nominal alpha: 0.05 Multiple Test correction: False Discovery Rate Keywords = SHIV89.6P Keywords = HIV Keywords = nonhuman primate Keywords = cDNA microarray Keywords = time course Keywords: time-course
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