Expression profiling of Gastric Cancers
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE2669 | GEO | 2005/07/25
SECONDARY ACCESSION(S): PRJNA92105
REPOSITORIES: GEO
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Project description:124 gastric biopsies comprising of Normals, Intestinal Metaplasia, Chronic Gastritis and Carcinomas are profiled. The ratio metric is given by (Biological sample / Universal Reference). The Biological Samples are labeled with Cy5 (Green) and the Pooled Normal Biopsies RNA is always labeled with Cy3 (Red). The arrays were scanned using a ScanArray 5000 scanner (Perkin-Elmer) and data extraction was done using Quantarray (GSI Lumonics). Subsequently, the data was imported into GeneSpring (Silicon Genetics) and intensity dependent LOWESS normalization was performed. The 7383 genes were filtered using an intensity in the reference channel (Cy3) of 300. This corresponded to 2 standard deviations above the mean for the background signal. Background singal was calculated using the empty wells/spots on the array and the average of 4 different positions on the slide were used.
2010-06-10 | E-GEOD-2669 | biostudies-arrayexpress
Project description:To identify Six1 target genes, we chose the mK4 cell line that represents later metanephric mesenchyme of the embryonic kidney. The cells were infected with adenovirus vectors (AxCAwt) overexpressing VP16-Six1wt or VP16-Six1W171R and cultured for 24h. Total RNA was extracted using Isogen reagent (Nippon Gene), treated with RNase-free DNase (Roche Diagnostics) and purified with RNeasy Midi Kit (QIAGEN). Poly A+ RNA was selected using Oligotex-dT30 latex beads (TaKaRa). Poly A+ RNA (20 µg) was reverse transcribed using an oligo(dT) primer in the presence of aminoallyl dUTP without the addition of spike-in controls. Then, single-stranded cDNAs were coupled with Cy3 (AxCAwt VP16-Six1wt infected sample) or Cy5 (AxCAwt VP16-Six1W171R infected sample) dyes, and the labeled probes were purified with QIAquick PCR Purification kit (QIAGEN). Hybridization to the microarray was carried out at 65°C for 17h according to the manufacturer's instruction. The arrays were washed, dried and scanned using ScanArray 5000 (GSI Lumonics) at two different PMT voltage settings (high and low) to avoid signal saturation. Cy3 and Cy5 intensities for each spot on the array were determined by QuantArray software (Version 3.0.0.0, GSI Lumonics). The raw data thus obtained was processed and the Cy3 to Cy5 ratios were calculated as follows: 1) subtraction of the fluorescence intensity of negative control spots as background from the intensity of each of the Cy3 and Cy5 spots, 2) normalization of the entire data set using the global normalization method, 3) elimination of spots with high background intensity for either dye, and 4) determination of the Cy3 to Cy5 ratios. To identify Six4 target genes, we chose the mK4 cell line that represents later metanephric mesenchyme of the embryonic kidney. The cells were infected with adenovirus vectors (AxCAwt) overexpressing VP16-Six4wt or VP16-Six4W263R and cultured for 24h. Total RNA was extracted using Isogen reagent (Nippon Gene), treated with RNase-free DNase (Roche Diagnostics) and purified with RNeasy Midi Kit (QIAGEN). Poly A+ RNA was selected using Oligotex-dT30 latex beads (TaKaRa). Poly A+ RNA (20 µg) was reverse transcribed using an oligo(dT) primer in the presence of aminoallyl dUTP without the addition of spike-in controls. Then, single-stranded cDNAs were coupled with Cy3 (AxCAwt VP16-Six4wt infected sample) or Cy5 (AxCAwt VP16-Six4W263R infected sample) dyes, and the labeled probes were purified with QIAquick PCR Purification kit (QIAGEN). Hybridization to the microarray was carried out at 65°C for 17h according to the manufacturer's instruction. The arrays were washed, dried and scanned using ScanArray 5000 (GSI Lumonics) at two different PMT voltage settings (high and low) to avoid signal saturation. Cy3 and Cy5 intensities for each spot on the array were determined by QuantArray software (Version 3.0.0.0, GSI Lumonics). The raw data thus obtained was processed and the Cy3 to Cy5 ratios were calculated as follows: 1) subtraction of the fluorescence intensity of negative control spots as background from the intensity of each of the Cy3 and Cy5 spots, 2) normalization of the entire data set using the global normalization method, 3) elimination of spots with high background intensity for either dye, and 4) determination of the Cy3 to Cy5 ratios. Keywords: other
2005-07-20 | GSE2043 | GEO
Project description:To identify Six1 target genes, we chose the mK4 cell line that represents later metanephric mesenchyme of the embryonic kidney. The cells were infected with adenovirus vectors (AxCAwt) overexpressing VP16-Six1wt or VP16-Six1W171R and cultured for 24h. Total RNA was extracted using Isogen reagent (Nippon Gene), treated with RNase-free DNase (Roche Diagnostics) and purified with RNeasy Midi Kit (QIAGEN). Poly A+ RNA was selected using Oligotex-dT30 latex beads (TaKaRa). Poly A+ RNA (20 µg) was reverse transcribed using an oligo(dT) primer in the presence of aminoallyl dUTP without the addition of spike-in controls. Then, single-stranded cDNAs were coupled with Cy3 (AxCAwt VP16-Six1wt infected sample) or Cy5 (AxCAwt VP16-Six1W171R infected sample) dyes, and the labeled probes were purified with QIAquick PCR Purification kit (QIAGEN). Hybridization to the microarray was carried out at 65°C for 17h according to the manufacturer's instruction. The arrays were washed, dried and scanned using ScanArray 5000 (GSI Lumonics) at two different PMT voltage settings (high and low) to avoid signal saturation. Cy3 and Cy5 intensities for each spot on the array were determined by QuantArray software (Version 3.0.0.0, GSI Lumonics). The raw data thus obtained was processed and the Cy3 to Cy5 ratios were calculated as follows: 1) subtraction of the fluorescence intensity of negative control spots as background from the intensity of each of the Cy3 and Cy5 spots, 2) normalization of the entire data set using the global normalization method, 3) elimination of spots with high background intensity for either dye, and 4) determination of the Cy3 to Cy5 ratios. To identify Six4 target genes, we chose the mK4 cell line that represents later metanephric mesenchyme of the embryonic kidney. The cells were infected with adenovirus vectors (AxCAwt) overexpressing VP16-Six4wt or VP16-Six4W263R and cultured for 24h. Total RNA was extracted using Isogen reagent (Nippon Gene), treated with RNase-free DNase (Roche Diagnostics) and purified with RNeasy Midi Kit (QIAGEN). Poly A+ RNA was selected using Oligotex-dT30 latex beads (TaKaRa). Poly A+ RNA (20 µg) was reverse transcribed using an oligo(dT) primer in the presence of aminoallyl dUTP without the addition of spike-in controls. Then, single-stranded cDNAs were coupled with Cy3 (AxCAwt VP16-Six4wt infected sample) or Cy5 (AxCAwt VP16-Six4W263R infected sample) dyes, and the labeled probes were purified with QIAquick PCR Purification kit (QIAGEN). Hybridization to the microarray was carried out at 65°C for 17h according to the manufacturer's instruction. The arrays were washed, dried and scanned using ScanArray 5000 (GSI Lumonics) at two different PMT voltage settings (high and low) to avoid signal saturation. Cy3 and Cy5 intensities for each spot on the array were determined by QuantArray software (Version 3.0.0.0, GSI Lumonics). The raw data thus obtained was processed and the Cy3 to Cy5 ratios were calculated as follows: 1) subtraction of the fluorescence intensity of negative control spots as background from the intensity of each of the Cy3 and Cy5 spots, 2) normalization of the entire data set using the global normalization method, 3) elimination of spots with high background intensity for either dye, and 4) determination of the Cy3 to Cy5 ratios.
2009-05-13 | E-GEOD-2043 | biostudies-arrayexpress
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2005-07-25 | GSE2637 | GEO
Project description:Examination of molecular basis of malaria resistance. Spleens from malaria resistant AcB55 and AcB61strains compared with A/J mice. Animals were sacrificed and spleens were rapidly frozen in liquid nitrogen, and subsequently stored at 80oC. Tissues were mechanically homogenized with a polytron and total cellular RNA was extracted using a commercially available reagent (TMTRIzol; Invitrogen). The poly-(A)+ fraction of the RNA was isolated by chromatography with oligo d(T) cellulose beads. For cDNA labeling, either 25 mg of total RNA or 2.5 mg of poly-(A) RNA was converted into cDNA using reverse transcriptase (RT; Super Script II, Invitrogen) and alternatively Cy5 or Cy3-labeled dCTP (1mM, Perkin Elmer-Cetus/NEN, Boston) in a reaction mixture containing 1.5 mL oligo (dT) (100pmol/ mL), 3 mL dNTP-dCTP (6.67mM each), 1 mL dCTP (2mM), 4 mL DTT (100mM), 8 mL 5 X RT Buffer (Invitrogen, California). The reactions were carried out at 42¼C for 3 hrs, after which the RNA was degraded by the addition of 0.5 mL RNase A (1 mg/mL) and 1.5 mL RNaseH 5 units/mL). Labeled cDNA was separated from unincorporated nucleotides (Qiagen column) and further concentrated by evaporation under vacuum. The arrays were pre-hybridized for 1-2hrs with DIGEasy hybridization buffer (Roche) containing 10ug/ml denatured salmon sperm DNA, and 10ug/ml yeast tRNA. The Cy5 and Cy3 labelled cDNAs were combined and hybridized in the same medium and incubated with the arrays for 16-18hrs at 37Co. Finally, the arrays were washed 3 x 10 mins in 0.1 x SSC (20 X SSC is 3M sodium chloride, 0.3 M sodium citrate, pH 7.0), 0.1%SDS at 50Co, and 4 x 3min in 0.1 x SSC at room temperature, and dried by centrifugation. Fluorescent array images were collected for both Cy3 and Cy5 with a ScanArray fluorescent scanner and image intensity data were extracted and analyzed with QuantArray 3.0 analysis software. Quantification data was imported in GeneSpring (Silicon Genetics). The import format was modified to maintain information relevant to user-contributed flagging as well as QC parameter furnished by the array manufacturer. Dye swap and Intensity-based normalization (Lowess) were performed using the default settings. Keywords: other
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2010-07-01 | E-GEOD-4495 | biostudies-arrayexpress
Project description:Microarray Expression profiles from 58 samples comprising of 3 normals and 55 gastric cancers is used for molecular subclassification of Gastric Cancers. Data Generation and Processing: The ratiometric data here is given by Ch1(Cy3)/Ch2(Cy5) where Ch2 is Universal Reference RNA from Stratagene. Two different chips were used (13K and 18k); The ratio metric is given by (Biological sample / Universal Reference). The Biological Samples are labeled with Cy3 (Green) and the Universal Reference RNA is always labeled with Cy5 (Red). Two types of spots were excluded a) Spots for which Foreground intensity was lower from background by lesser than 10 fluoresence units. b) Spots that couldnt be identified reliably by the scanner (Arrayworx) software within the "region of interest" masked by the user. The 2 channels for each array are equalized by multiplying the reference channel Ch2(Cy5) by a constant which is derived by (Total Cy3 / Total Cy5). Subsequently, all expression ratios from each array is normalized to have median expression value = 1.
2010-06-09 | E-GEOD-2637 | biostudies-arrayexpress
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2010-10-08 | E-GEOD-8502 | biostudies-arrayexpress
Project description:A total of 20 KYSE human esophageal squamous cell carcinoma cell lines (KYSE-30, -140, -150, -170, -180, -200, -220, -350, -410, -450, -510, -520, -590, -770, -850, -890, -1170, -1190, -1250, and -2270) and a non-cancerous esophageal epithelial cell (HEEC-1)were kindly provided by Dr. Y. Shimada (Kyoto University, Kyoto, Japan). The KYSE cell lines were cultured in RPMI 1640 medium (Life Technologies, Inc., Grand Island, NY) containing 10% heat-inactivated fetal bovine serum (FBS; BioWhittaker, Verviers, Belgium) in a humidified atmosphere of 5% CO2 and maintained in continuous exponential growth by passage every 3 days. HEEC-1 cells were cultured in Keratinocyte SFM medium with growth supplement containing 2.5 mg EGF and 25 mg bovine pituitary extract in 500 mL liquid basal medium (GIBCO BRL, Rockville, MD) and expanded by passage twice in a week. The exponentially growing cultured cells (2 x 10^6) were collected after two-washings with PBS. Total RNA was prepared from the frozen cell pellets using QIAGEN RNeasy mini kit (QIAGEN, Inc., Valencia, CA), and mRNA was purified using MACS mRNA Isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the supplier’s protocols. RIKEN human 21K array containing 20,784 clones with positive and negative controls was used to analyze gene expression of 20 KYSE esophageal cancer cell lines using HEEC-1 as reference. The target DNAs used to construct human 21 K array were the glycerol stock of cDNA clones purchased from ResGen (Invitrogen Corporation, Carlsbad, CA)). One-microgram poly(A) RNAs from KYSE cells and the reference cells were labeled with Cy3-dCTP and Cy5-dCTP, respectively, by random-primed reverse transcription. Arrays were laser-scanned using ScanArray 5000TM confocal laser scanner (GSI Lumonics, Billerica, MA), and the images were analyzed using ScanAlyzeTM (Stanford University). All experiments were performed in duplicate (_1 & _2). Keywords: parallel sample
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