Ambrosino series 1
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE1568 | GEO | 2004/12/01
SECONDARY ACCESSION(S): PRJNA90887
REPOSITORIES: GEO
Similar Datasets
Project description:To characterize the properties and evaluate the performance of the TAcKLE procedure, a novel method providing effective transcriptome amplification for expression analyses on oligonucleotide microarrays, we performed 20 two-color hybridizations using self-spotted Operon 27k arrays. A single source of universal human reference RNA (pooled from 10 cell lines) and RNA extracted from healthy breast tissue was used for all experiments to avoid differences in transcript abundance imposed by the RNA preparation. RNA was either amplified by one or two rounds of linear isothermal RNA amplification (starting from 2000 ng, 200 ng, 20 ng or 2 ng), followed by Cy-dUTP incorporation using Klenow fragment, or labeled directly by reverse transcription of 40 µg RNA with Cy-dUTP. For all quantities of starting material, one co-hybridization of reference RNA, two hybridizations of breast versus reference RNA as well as one hybridization of reference versus breast RNA were performed. In case of amplified RNA, all dye labeling reactions were made from distinct target preparations. Hybridizations were performed for 16 h at 42 °C in a GeneTAC Hybridization Station (Genomic Solutions) using UltraHyb hybridization buffer (Ambion). Hybridized microarrays were scanned at 5 µm resolution on a GenePix 4000B microarray scanner (Axon Instruments). For all hybridizations, raw signal intensities were normalized applying variance stabilization (W. Huber et al., Bioinformatics 18 Suppl 1, 2002). Keywords = amplification Keywords = labeling Keywords = protocol Keywords = spotted Keywords = human Keywords = long oligonucleotide Keywords = oligo Keywords = 70mer Keywords = Operon Keywords: other
2004-08-13 | GSE1645 | GEO
Project description:To characterize the properties and evaluate the performance of the TAcKLE procedure, a novel method providing effective transcriptome amplification for expression analyses on oligonucleotide microarrays, we performed 20 two-color hybridizations using self-spotted Operon 27k arrays. A single source of universal human reference RNA (pooled from 10 cell lines) and RNA extracted from healthy breast tissue was used for all experiments to avoid differences in transcript abundance imposed by the RNA preparation. RNA was either amplified by one or two rounds of linear isothermal RNA amplification (starting from 2000 ng, 200 ng, 20 ng or 2 ng), followed by Cy-dUTP incorporation using Klenow fragment, or labeled directly by reverse transcription of 40 µg RNA with Cy-dUTP. For all quantities of starting material, one co-hybridization of reference RNA, two hybridizations of breast versus reference RNA as well as one hybridization of reference versus breast RNA were performed. In case of amplified RNA, all dye labeling reactions were made from distinct target preparations. Hybridizations were performed for 16 h at 42 °C in a GeneTAC Hybridization Station (Genomic Solutions) using UltraHyb hybridization buffer (Ambion). Hybridized microarrays were scanned at 5 µm resolution on a GenePix 4000B microarray scanner (Axon Instruments). For all hybridizations, raw signal intensities were normalized applying variance stabilization (W. Huber et al., Bioinformatics 18 Suppl 1, 2002). Keywords = amplification Keywords = labeling Keywords = protocol Keywords = spotted Keywords = human Keywords = long oligonucleotide Keywords = oligo Keywords = 70mer Keywords = Operon Keywords: other
2004-08-13 | E-GEOD-1645 | biostudies-arrayexpress
Project description:Experiment description 1. Experimental design 1) Authors, laboratory, and contact: Xue-Lin Cui1, Patricia Soteropoulos2, Peter Tolias2, Ronaldo P. Ferraris1: 1Dept. of Pharmacology and Physiology, UMDNJ-New Jersey Medical School, Newark, NJ 07103-2714, 2Center for Applied Genomics, PHRI, 225 Warren Street Newark, NJ 07103-3506. Contact to Dr. Ronaldo P. Ferraris. 2) Type of the experiment: treated vs. untreated comparison. 3) Experimental factors: Twenty to twenty-two old SD rats were use in the experiment. The intestines of paired pups were perfused with 100 mM fructose (HF) and 100 mM glucose (HG) for 4-h or 20-min, respectively. 4) Totally 14 experiments were done. 5) A common reference was not used for all the hybridizations. 6) Quality control steps ( 14) Miller RA, Galecki A, Shmookler-Reis RJ. Interpretation, design, and analysis of gene array expression experiments. J Gerontol A Biol Sci Med Sci. 2001 Feb;56(2):B52-7. (15) Tseng GC, Oh MK, Rohlin L, Liao JC, Wong WH. Issues in cDNA microarray analysis: quality filtering, channel normalization, models of variations and assessment of gene effects. Nucleic Acids Res. 2001 Jun 15;29(12):2549-57. ( 16) Yang YH, Speed T. Design issues for cDNA microarray experiments. Nat Rev Genet. 2002 Aug;3(8):579-88. (17) Yang YH, Dudoit S, Luu P, Lin DM, Peng V, Ngai J, Speed TP. Normalization for cDNA microarray data: a robust composite method addressing single and multiple slide systematic variation. Nucleic Acids Res. 2002 Feb 15;30(4):e15.): a) In the 4 h perfusion experiment, the intestines of the paired pups were perfused with 100 mM fructose (HF) and 100 mM glucose (HG) for 4-h, respectively. Totally five replicates were conducted. The RNA samples were extracted from HF- and HG-perfused intestines (n=5 pairs) then labelled with Cy 5 (HF1, HF2, HF3, HF4, and HF5) and Cy 3 (HG1, HG2, HG3, HG4, and HG5), respectively. At the same time, one dye swap experiment, in which the samples extracted from HF- and HG-perfused intestines were labelled with Cy 3 (HF1) and Cy 5 (HG1), respectively, was done and used in the data analysis. b) In the 20 min perfusion experiment, the intestines of of the paired pups were perfused with 100 mM fructose (HF) and 100 mM glucose (HG) for 20-min, respectively. Totally four replicates were conducted. In two of the four-replicate experiments, the samples extracted from HF- and HG-perfused intestines were labelled with Cy 5 (HF1a and HF3a) and Cy 3 (HG1a and HG3a), respectively. In the other two of the four-replicate experiments, the dye was swapped, that is, the samples extracted from HF- and HG-perfused intestines were labelled with Cy 3 (HF2a and HF4a) and Cy 5 (HG2a and HG4a), respectively. c) Three self-self experiments using different samples (HF1, HG1, and HF2) and one capture primer alone experiment were conducted to eliminate the false positive genes and aid to analyze the data. 7) The goal of the experiment: Fructose induced the expression of GLUT5 mRNA in the small intestinal epithelial cells. But it is not clear about the mechanisms involved in the phenomenal. In this experiment, we hypothesize: the expression of some genes must be changed after fructose perfusion for 4-h, these genes may regulate GLUT5 gene transcription; the expression of some genes must be changed even after 20-min fructose perfusion, these genes may trigger the GLUT5 gene expression as early response gene. Our goal in this study is to find out these potencial gene. 8) Links (URL), citations 2. Samples used, extract preparation, labeling and hybridization 1) Bio-source properties a) Sprague Dawley rats (NCBI taxonomy); b) The rat was anesthetized using urathene. The rat body was kept at 37 oC under light. c) Descriptors relevant to the particular sample: the jejunum of the 20-d old rat pups (unknown sex) was perused. 2) Biomaterial manipulations: the jejunum was in vivo perfused with 100 mM HF or HG for 4-h or 20-min. At the end of each experiment, the jejunum was immediately cut out and snap frozen in liqued nitrogen, and then stored the tissue in -80 oC freezer. 3) RNA isolation: A RNeasy Midi Kit (QIAGEN Inc., Valencia, CA) was used to extract total RNA from the jejunum. The brief procesure is described in the Total RNA Extraction for RT-PCR and Microarray above. 4) Sample assignment to slides for labeling and hybridization: S1: HF1(Cy 5) vs HG1 (Cy 3); S2: HF2 (Cy 5) vs HG2 (Cy 3); S3: HF3 (Cy 5) vs HG3 (Cy 3); S4: HF4 (Cy 5) vs HG4 (Cy 3); S5: HF5 (Cy 5) vs HG5 (Cy 3); S6: HF2 (Cy 3) vs HG2 (Cy 5); S7: HF1a (Cy 5) vs HG1a (Cy 3); S8: HF3a (Cy 5) vs HG3a (Cy 3); S9: HF2a (Cy 3) vs HG2a (Cy 5); S10: HF4a (Cy 3) vs HG4a (Cy 5); S11: HG1 (Cy 5) vs HG1 (Cy 3); S12: HG3 (Cy 5) vs HG3 (Cy 3); S13: HF3 (Cy 5) vs HF3 (Cy 3); S14: Cy 3 vs Cy 5 capture primer. 5) Labeling and hybridization procedures and parameters: The 3DNA Submicro Oligo Expression Array Detection Kit (Genisphere Inc., Hatfield, PA) was employed to synthesize and label the complimentary DNA (cDNA) with fluorescent dye: Cy3 or Cy5. The reason why this indirect labeling method was chosen is based on a previous study which demonstrated that the method was more sensitive and consistent than direct labeling method or other indirect techniques like aminoallyl labeling method and MICROMAX tyramide signal amplification labeling method. (18)Yu J, Othman MI, Farjo R, et al: Molecular Vision 2002; 8:130-137). In the primary experiments, three, four, and five g of total RNA were tested for labeling and hybridization conditions. In the official experiments, four g of total RNA isolated from each sample were used for labeling and hybridization, because the signal to noise ratio is high at this concentration. In addition, at this concentration, all most of the low expressed genes are detectable and the highly expressed genes are not fully saturated. The procedure was conducted following the manufacturer's protocol. Briefly, the first strand cDNA was synthesized using a specific poly T Cy3 RT primer or a specific poly T Cy5 RT primer in different tubes for paired samples. Combine the Cy3 and Cy5 reactions in one tube and precipitate the sample in ethanol. After briefly drying the pellet, resuspend the pellet with 40 l of hybridization buffer. Subsequently, apply the hybridization mixture onto the array slide and hybridize overnight at 55 oC in a humidified chamber. Following the hybridization, the slides were washed by placing them into 50 ml conical tubes containing 2 x SSC, 0.2% SDS for 15 min, 2 x SSC for 10 min, 0.2 x SSC for 10 min, and 95% ethanol for 1 min all at room temperature, except for the first wash, in which the temperature was 42 oC. After drying the slides, the Cy3 and Cy5 capture (connected to dye molecues) reagents mixed in hybridization buffer was pipeted onto the slides, and then hybridize at 65 oC for 3 h following slides wash as described above. 3. Measurement data and spesifications of data processing 1) Raw data description Slides were scanned for Cy3 and Cy5 fluorescence using an Axon GenePix4000 microarray reader (GenePix 4000A, Axon instruments, Foster City, CA) and quantitated using GenePix software. Scan parameters: Scan power: Cy 3 : Cy 5 = 100:100; Laser power: Cy 3 : Cy 5 = 1.15:1.76; Spatial resolution: ???; Pixel size: 10 or 100 ???; Pixel number: F pixels: 50-100; B pixels: 400-500 ???; PMT voltage: 600-750 for Cy 5 channel, 650-850 for Cy 3 channel. 2) Image analysis and quantitation For the image analysis, the density of the spots was detected using Gene Spring (Silicon Genetics, Redwood City, CA) software (GeniPix 4.0) (19) Axon Instruments Inc. (1999) GenePix 4000A User's Guide. Axon Instruments, Union City, CA). It is commercially available. Description of the algorithm and all the parameters used: R = red channel signal = F635; G = green channel signal = B 532; M = log2(R/G); A = 1/2xlog2(RxG); User-difined parameter f = 40%. For the complete image analysis out put of each image, see the NCBI's GEO database (www.ncbi.nlm.nih.gov/geo). 3) Normalized and summarized data-gene expression data matrix For the normalization, the following two steps were taken. a) When measuring the density of the spots using Gene Spring software, the ratio of Cy 3 and Cy 5 dyes was adjusted as 1 as a globle normalization for the whole slide. b) For the gene expression analysis, the results (midian of the density of the pixel in one spot was used) were normalized by a LOWESS (locally weighed scatter smoother) software provided by the Center for Applied Genomics, so that the results can be compared among the experiments performed at different times. (20)Yang YH, Dudoit S, Luu P, Lin DM, Peng V, Ngai J, Speed TP. Normalization for cDNA microarray data: a robust composite method addressing single and multiple slide systematic variation. Nucleic Acids Res. 2002 Feb 15;30(4):e15.). c) The following rules were made to decide the genes that were significantly changed: 1) Eliminate the genes with high density (over than 2X of background) in the 3DNA capture probe experiments; 2) Eliminate the genes with density lower than 2X of background ( 21) Lee HM, Greeley GH Jr, Englander EW. Age-associated changes in gene expression patterns in the duodenum and colon of rats. Mech Ageing Dev. 2001 Apr 15;122(4):355-71.); 3) Eliminate the genes with change over than 1.5X in dye flip-over experiment; 4) Eliminate the genes with opposite results in one experiments between dye flip-over comparisons; 5) Only the genes significantly changed over 3 in 5 comparisons in 4 h perfusion experiment and over 2 in 4 comparisons in 20 min perfusion experiment were considered as reliable and biologically important. d) The final results discribed in this paper were presented as Means + SE and the frequency of gene changed in the HF-perfused intestine. About the image, raw, and normalized data, and other detailed parameters, please see the gene expression data tables derived from the experiments in the NCBI's GEO database. The design for the dye labeling in this experiments is as the follows: 1) 5 comparisons were conducted in the 4 h-perfused HG vs HF experiment, in which in 3 comparisons, the cDNA samples synthesized from HG- and HF-perfused intestines were labeled with Cy3 and Cy5, respectively; and in 2 comparisons, the cDNA samples synthesized from HG- and HF-perfused intestines were labeled with Cy5 and Cy3, respectively. 2) 4 comparisons were conducted in the 20 min-perfused HG vs HF experiment, in which in 2 comparisons, the cDNA samples synthesized from HG- and HF-perfused intestines were labeled with Cy3 and Cy5, respectively; and in 2 comparisons, the cDNA samples synthesized from HG- and HF-perfused intestines were labeled with Cy5 and Cy3, respectively. 3) 3 dye flip-over experiments using 1 RNA sample extracted from HF-perfused intestine and 2 samples extracted from HG-perfused intestines were performed to eliminate the false positive results induced by the dye labeling technique or other unsolved problems. 4) 2 experiments only applying 3DNA fluorescent probe, which is connected to complementary capture sequence, were performed to eliminate the false positive genes that were low expressed in the tissue. The procedures for synthesizing the first strand cDNA, labeling the cDNA, hybridizaion, and detecting the signals were performed following the manufacturer's protocols. Keywords: other
2004-01-28 | GSE996 | GEO
Project description:This series represents the effects of OVA, tg-IL-13, and direct effects of IL-13 on airway epithelial cells. Experiment Design: Type of experiment: comparison of three related murine models of asthma, 1) Ova model, 2) tg-IL-13 model, 3) IL-13/Epi model. Two control groups were used: PBS challenged mice to control for the effects of OVA and tg-IL-13+ and STAT6-/- mice as controls for tg-IL-13 and IL-13/Epi mice. Measurements were made in each model in two types of tissue samples, whole lung and tracheal perfusate. Experimental factors: Allergen vs sham challenge, natural STAT6 expression vs transgenic expression and no expression. The number of hybridizations performed in the experiment: 50 (25 whole lung and 25 tracheal perfusate). The type of reference used for the hybridizations, if any: Pooled reference from lung (whole lung or tracheal perfusate) from untreated wildtype mice. Hybridization design: two-color hybridizations with reference design. Each individual sample was hybridized to a separate array. Quality control steps taken: 5 experimental replicates per group. RNA integrity assessed by Agilent Bioanalyzer. Arrays with low signals, high background, or high spatial variation rejected and corresponding samples reanalyzed. URL of any supplemental websites or database accession numbers:GEO (http://www.ncbi.nlm.nih.gov/geo, accession number GSE1438). ********** Samples used, extract preparation and labeling: The origin of the biological sample: homogenized whole lung from mouse and perfusate of mouse trachea. Manipulation of biological samples and protocols used: Whole lungs mechanically homogenized in 7.0 ml Trizol reagent (Invitrogen) for total RNA collection. Tracheas perfused with lysis buffer (Qiagen Rneasy kit) for total RNA collection. Protocol for preparing the hybridization extract and labeling: Preparation of aminoallyl-UTP labeled whole lung cDNA was performed as described [1]. Total RNA from tracheal perfusate (1.0 - 1.5 g per sample) was amplified and labeled with aminoallyl-dUTP using the MessageAmp aRNA kit (Ambion). Labeled cRNAs were coupled to Cy3 or Cy5 dyes (CyScribe, Amersham Biosciences) and purified as described [1]. Labeling protocol(s): Coupled to Cy3 or Cy5.External controls (spikes): none. ********** Hybridization procedures and parameters: Hybridizations were performed as described [1] except Ambion SlideHyb Glass Array Hybridization Buffer #1 (neat concentration) was used as hybridization buffer and hybridizations were carried out for 40 h at 55 °C. Following hybridization, arrays were washed in 1X SSC with 0.03% SDS (wash 1), 0.2X SSC (wash 2), and 0.05X SSC (wash 3) for five minutes for each wash. ********** Measurement data and specifications: Arrays were scanned using an Axon Genepix 4000B scanner and GenePix Pro Analysis 5.0 software; laser power 100%, 10 micron resolution, PMT optimized for each array. Data from GPR files are available from GEO (http://www.ncbi.nlm.nih.gov/geo, accession number GSE1438).The “print-tip loess” normalization was used to correct for within-array dye and spatial effects and single channel quantile normalization was used to facilitate comparison between arrays. No background subtraction was performed. We used functions in the library marrayNorm of the R / Bioconductor package to perform these normalizations. After normalization we determined the log2 ratio of experimental sample intensity to reference sample intensity for each probe on each array. ********** Array Design: General array design Array design name: UCSF 10Mm Mouse v.2 Oligo Array (GEO GPL1089) and UCSF Gladstone 18K Mouse v.2 Oligo Array (GPL1196) Platform type: spotted oligonucleotides (70mers) Surface and coating specification: aminosilane coated glass slides Physical dimensions of array support (e.g. of slide): 25 x 75 x 1.0 mm Number of features on the array: GPL1089: 19152, GPL1196: 18240 (including empty and duplicate features) For production protocol, see http://www.microarrays.org/pdfs/PrintingArrays.pdf Feature information is available from GEO (GSE1438). Reporters: synthetic single stranded oligonucleotides. Sequence and annotation information available from GEO (GPL1089 and GPL1196) Method of reporter preparation: synthesized by Operon. The spotting protocols used, including the array substrate, the spotting buffer, and any post-printing processing, including cross-linking: see http://www.microarrays.org/pdfs/PrintingArrays.pdfAny additional treatment performed prior to hybridization: none. ********** Reference: 1. Barczak A, Rodriguez MW, Hanspers K, Koth LL, Tai YC, et al. (2003) Spotted long oligonucleotide arrays for human gene expression analysis. Genome Res 13: 1775-1785. Keywords = IL-13 Keywords = Epithelial Keywords = differential gene expression Keywords = microarray Keywords: parallel sample
2005-05-27 | GSE1438 | GEO
Project description:HEK293 cells were transfected with control plasmid (pcDNAI/Neo;Invitrogen) or with the plasmid encoding HCaRG. Stable transfectants were synchronized and grown in the presence of 10% FBS for 48 h. Total RNAs were purified with the mini RNeasy kit (Qiagen). Chips HG-U133A: - Labeling protocol: Enzo BioArray HighYield RNA Transcript Labeling Kit. - Hybridization: According to the manufacturer's protocol (Affymetrix). - Scanner: GeneArray 2500. - Normalization: Employing GCOS software, chips were normalized using all probe sets scaling option and target signal at 500. Chips HG-U133 Plus 2.0: - Labeling protocol: GeneChip IVT Labeling Kit. - Hybridization: According to the manufacturer's protocol (Affymetrix). - Scanner: GeneChip Scanner 3000. - Normalization: Employing GCOS software, chips were normalized using all probe sets scaling option and target signal at 500. Keywords: parallel sample
2005-10-19 | GSE2555 | GEO
Project description:Eight “self-self” hybridizations were prepared as follows. Total RNA was isolated from each of three rat livers and brains, and equal mass amounts of each of these six samples were combined to create a mixed RNA population. Cy3- and Cy5-labeled cDNA was generated from this RNA mixture using an aminoallyl nucleoside analog indirect labeling protocol. Specifically, eight separate 20 microgram aliquots of the total RNA mixture were reverse transcribed into cDNA in the presence of aminoallyl-dUTP and the resulting cDNA was then coupled to either Cy3 or Cy5 mono NHS ester (four reactions with each label). Keywords: Self-Self Hybridization
2007-09-11 | GSE8950 | GEO
Project description:NT2 cells were treated with DMSO (control) or 10uM Retinoic Acid (RA). Total RNA was isolated using TriReagent, and subsequent cleanup with RNeasy columns (Qiagen) according to the manufacturer's directions. Hybridizations were performed according to Affymetrix guidelines using the Human HG-U133A chip containing 22,500 unique genetic elements. Double-stranded cDNA was synthesized using Superscript II RT (Invitrogen). In vitro transcription labeling was performed with biotin labeled nucleotides using the Bioarray High Yield RNA Labeling Kit (ENZO). 20 ul of labeled RNA per sample was added to the hybridization cocktail, with a total of 15 ug hybridized to the chip (16 hours, 45C). RMA analysis was performed using Gene Traffic software (Iobion), where raw data for each hybridization was normalized, background corrected and filtered for signal intensity of 50 or greater in at least one comparison. Keywords: other
2005-10-18 | GSE2448 | GEO
Project description:Both spotted long oligonucleotide arrays (GPL1384) and Affymetrix GeneChip arrays (GPL96) were used to analyze gene expression in six human head and neck squamous cell carinoma samples versus control samples or lymph node metastases of the same patients. Hybridizations of HG-U133A GeneChip arrays were performed using standard Affymetrix protocols and equipment. Before hybridization on DKFZ Operon 27k long oligonucleotide arrays, 2 µg RNA were amplified by one round of linear isothermal RNA amplification, followed by Cy-dUTP incorporation using Klenow fragment. Hybridizations were performed for 16 h at 42 °C in a GeneTAC Hybridization Station (Genomic Solutions) using UltraHyb hybridization buffer (Ambion). Hybridized microarrays were scanned at 5 µm resolution on a GenePix 4000B microarray scanner (Axon Instruments). Raw signal intensities from both platforms were normalized applying variance stabilization (W. Huber et al., Bioinformatics 18 Suppl 1, 2002). Expression ratios were compared for those genes represented in both array platforms. Keywords: parallel sample
2005-03-01 | GSE1722 | GEO
Project description:Total RNA from each sample was quantified by the NanoDrop ND-1000 and RNA integrity was assessed by standard denaturing agarose gel electrophoresis. Total RNA of each sample was used for labeling and array hybridization as the following steps: 1) Reverse transcription with by Invitrogen Superscript ds-cDNA synthesis kit; 2) ds-cDNA labeling with NimbleGen one-color DNA labeling kit; 3) Array hybridization using the NimbleGen Hybridization System and followed by washing with the NimbleGen wash buffer kit; 4) Array scanning using the Axon GenePix 4000B microarray scanner (Molecular Devices Corporation). Scanned images (TIFF format) were then imported into NimbleScan software (version 2.5) for grid alignment and expression data analysis. Expression data were normalized through quantile normalization and the Robust Multichip Average (RMA) algorithm included in the NimbleScan software. The Probe level (*_norm_RMA.pair) files and Gene level (*_RMA.calls) files were generated after normalization. The 2 gene level files were imported into Agilent GeneSpring GX software (version 11.5.1). Genes that have values greater than or equal to lower cut-off: 100.0 in that at least 1 out of 2 samples (“All Targets Value”) were chosen for data analysis.
2022-07-20 | GSE208583 | GEO
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
2004-11-03 | GSE1854 | GEO