TAcKLE - Transcriptome Amplification and cDNA Labeling for Expression analysis
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE1645 | GEO | 2004/08/13
SECONDARY ACCESSION(S): PRJNA90697
REPOSITORIES: GEO
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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: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:Hybridizations were performed in triplicate, using total of 7 RNA samples extracted from 3 independent batches of proliferating cardiomyocyte cell line derived from p38alpha knockout mouse. In two hybridizations (Ambrosino 1, Ambrosino 2), the RNAs from p38alpha-/- and wt cells were labeled with Cy-5 and Cy-3-dUTP, respectively, whereas in the third hybridization (Ambrosino 3), the dyes were swapped. The multiple spike-in control RNA templates were added to the sample upon direct labeling, and the successful labeling and hybridization was confirmed in each hybridizations. Twelve housekeeping genes in the array (GAPDH, HPRT and S16, S9, and S8 ribosomal proteins) gave average value of Ratio of Medians (ROM), 1.30 ± 0.26, which was confirmed to be acceptable. GenePix Pro program calculates the Normalization Factor, based on the premise that the arithmetic mean of the ratios from every feature on the given array should be equal to 1. Normalization was therefore performed by multiplying the Factor to ROM in each gene. The program also identifies features that did not give good alignment to the expected spotted area as “flagged” spots, indicative of the impaired-quality hybridization of the specific genes. All flagged genes were removed from the list before further analysis. The genes that passed all these criteria were sorted by ROM and those that showed more than ±1.5 fold changes were selected in the data table from each hybridization. Finally, the data tables from the 3 independent hybridizations were compared and only those genes that appeared in all 3 experiments were selected in the final list (manuscript submitted). Keywords = NIA mouse 15K Keywords = cardiomyocytes Keywords = p38alpha MAPK Keywords: repeat sample
2004-12-01 | GSE1568 | GEO
Project description:Both spotted long oligonucleotide arrays and Affymetrix GeneChips were used to measure differential gene expression in two RNA samples (K562 erythroleukemia RNA and the Stratagene Universal Human Reference RNA). For Affymetrix technology, the two RNAs were analyzed separately. There are two replicates for K562 RNA (GSM4843 and GSM4844) and three for the Stratagene Universal reference (GSM4845-GSM4847). Two types of spotted long oligonucleotide arrays were used. These arrays were used to do two-color hybridizations to directly compare K562 and Universal reference RNAs. One array (GPL273) was made with probes from the Operon Human Genome Oligo Set Version 1. These arrays were used with unamplified cDNA probes (6 replicates, GSM4848-GSM4853), with aRNA probes produced by one round of T7 RNA polymerase-based amplification (6 replicates, GSM4854-GSM4859), and with aRNA probes produced by two rounds of amplification (2 replicates, GSM4860-GSM4861). Keywords: parallel sample
2003-03-18 | GSE344 | GEO
Project description:NIH3T3 cells were cultured in medium containing 1 uM, 100 nM, 10 nM and 0 nM (serve as control) shld1 for 24 hours. Three independent cultures were obtained for each drug concentration. Total RNA from experimental samples were extracted using RNeasy Kit (Qiagen). Mouse RNA pooled from 11 cell lines (Stratagene, La Jolla, CA) was used as reference. Cy5-dUTP and Cy3-dUTP, were used to label the experimental cDNA and the reference samples, respectively, and hybridized to the Mouse Exonic Evidence Based Oligonucleotide (MEEBO) arrays (http://www.microarray.org./sfgf/meebo). The hybridizations were performed by Stanford Functional Genome Facility using its standard protocol (Oligo Array Hybridization protocol, http://www.microarray.org./sfgf/docView.do?type=2). A compound treatment design type is where the response to administration of a compound or chemical (including biological compounds such as hormones) is assayed. Keywords: compound_treatment_design
2007-09-27 | GSE5916 | GEO
Project description:We completed genome-wide microarray analysis in Sirt6 knockout (ko) mouse embryonic fibroblasts (MEFs) and control wildtype (wt) MEFs. Prior to RNA extraction, MEFs were treated with TNF-alpha (10 ng/ml) for the indicated times. Total RNA was extracted with TRIzol (Invitrogen) and amplified with Ambion Amino Allyl MessageAmp II Amplification kit. Amplified mouse Universal Reference RNA (Stratagene) was used as reference RNA for analysis. Array hybridization of mouse MEEBO arrays is as described (Hendrickson et al., PLoS ONE, 2008). Groups of assays that are related as part of a time series. Compound Based Treatment: 10 ng/ml TNF-alpha Keywords: time_series_design
2009-01-09 | GSE13207 | GEO
Project description:We established HeLa cell lines stably expressing a short hairpin RNA against SIRT6 (S6 sh2), and then completed genome-wide microarray analysis in shSIRT6 knock-down and control pSR HeLa cells. Prior to RNA extraction, HeLa cells were treated with TNF-alpha (5 ng/ml) for the indicated times. Total RNA was extracted with TRIzol (Invitrogen) and amplified with Ambion MessageAmp RNA Amplification kit. Amplified human Universal Reference RNA (Stratagene) was used as reference RNA for analysis. Construction and array hybridization of human cDNA microarrays were performed as previously described (Perou et al., 2000). Groups of assays that are related as part of a time series. Compound Based Treatment: 5 ng/ml TNF-alpha Keywords: time_series_design
2009-01-09 | GSE13206 | GEO
Project description:Each strain was grown overnight then diluted in fresh rich media. After three hours strains were again rediluted into either rich media or rich media supplemented with copper sulfate. After another three hours cultures were sampled, cells lysed and flash frozen using liquid nitrogen. RNA was extrated using hot phenol-chloroform, reverse transcripbed using amino-allyle dUTP and labelled with either Cy3 or Cy5 flourescent dye. Each hybridization is of a single sample compared to a reference pool contructed from all the strains with the same treatment. Labelled probe were hybridized to DNA microarrays spotted with 6144 70 bp oligonucleotides obtained from Qiagen-Operon. After an overnight hybridization, microarrays were scanned using a GenePix 4000A scanner and spot intensities extracted using GenePix 4.0 software. Bad spots were flagged based on the image. Keywords: other
2004-03-01 | GSE1073 | 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:As part of a larger conifer genomics project, we developed a new 21.8K cDNA microarray containing 21,843 spotted ESTs from spruce (Picea spp.). To evaluate the performance of the array we conducted four technical replicate self-self hybridizations using untreated control bark RNA pooled from four biological replicates. The objectives were to determine: 1) the linear dynamic range of expression detection of the array; 2) the level of non-specific hybridization to negative control elements on the array; and 3) the overall technical reproducibility of the array. Keywords: Array performance evaluation using self-self hybridizations.
2008-06-01 | GSE8363 | GEO