Gene Expression Profiling of human leukemic B-cells. Two-color Microarray-Based Gene Expression Analysis.
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE68094 | GEO | 2015/04/22
SECONDARY ACCESSION(S): PRJNA281734
REPOSITORIES: GEO
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Project description:Fresh MEC1 cells (chronic lymphocytic leukemia) (ACC-497, DSMZ) previously untreated and treated in cultures in vitro with glycodendrimers (nanoparticle with maltotriose on the surface). MEC1 cells were centrifuged after incubation with drugs or dendrimers. Agilent’s Low Input Quick Amp Labeling Kit was used to generate fluorescent cRNA (complimentary RNA) with a sample input 100ng of total RNA for two-color processing. The method uses T7 RNA polymerase, which simultaneously amplifies target material and incorporates cyanine 3- or cyanine 5-labeled CTP. Amplification is typically at least a 100-fold from total RNA to cRNA with the use of this kit. cRNA quantification had been done using NanoDrop ND-1000 UV-VIS Spectrophotometer version 3.2.1. Arrays were placed onto the gaskets and than to the slide chamber in rotisserie in a hybridization oven set. Hybrydization was carried out at 65-Celsius degrees for 17 hours. Arrays were scanning using Agilent Scanner. Scan resolution was set up for 3μM. Microarray gene expression profiling. Intensity of gene expression in MEC1 cells before treatment was compared with the gene expression signals in MEC1 cells from the same cell culture after 4 and 48 hours of treatment. MEC1 cells were treated with PPI-G4-OS-Mal-III dendrimer, PPI-G4-DS-Mal-III, Fludarabina. Sample titles represent; A = Cy3 B = Cy5 R = reference (48h incubation) R4 = reference (4h incubation) 1 = PPI-G4-DS-Mal III (1st cell culture flask) (48h incubation) 2 = PPI-G4-DS-Mal III (2nd cell culture flask)(48h incubation) 3 = PPI-G4-OS-Mal III (1st cell culture flask)(48h incubation) 4 = PPI-G4-OS-Mal III (2nd cell culture flask)(48h incubation) 5 = PPI-G4-DS-Mal III (4h incubation) 6 = PPI-G4-OS-Mal III (4h incubation) 7 = FA (4h incubation) 8 = FA (48h incubation)
2015-04-22 | E-GEOD-68094 | biostudies-arrayexpress
Project description:Capacity of exercise and performance is the most valuable in the horses. They have been selected for strength, speed, and indurance trait. Athletic pheno types are influenced markedly by environment, management, and training. However, it has long been accepted that there are underlying genetic factors. To determine altered mRNA expression in circulating leukocytes of horses induced by exercise. Healthy neutered male warmblood horses were subjected to indoor exercise (trotting with alternative cantering for 6o minutes). Peripheral blood was collected from the jugular vein before and after the exercise, and subsequently buffy coat leukocytes were isolated by centrifugation. Total RNAs was isolated. Cyanine 3-labeled cRNA (complementary RNA) was generated from Agilent’s Low RNA Input Linear Amplification kit with 500 ng total RNA. Labeled cRNA was applied microarray (Agilent technologies, 8x60K) using Agilent’s Gene Expression Hybridization Kit. The present study revealed a subset of mRNAs in equine peripheral blood leukocytes affected by exercise, providing background information for genes associated with exercise in warm-blood horses.
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Project description:Spleen total RNA (Catalog #64044-1) and liver total RNA (Catalog #64042-1) were purchased from Clontech and used as the starting material. Spleen was labeled with Cyanine-3 (Green) while liver was labeled with Cyanine-5 (Red). cRNA was made by the single round amplification method. Keywords: repeat sample
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Project description:Spleen total RNA (Catalog #64044-1) and liver total RNA (Catalog #64042-1) were purchased from Clontech and used as the starting material. Spleen was labeled with Cyanine-3 (Green) while liver was labeled with Cyanine-5 (Red). cRNA was made by the double round amplification method. Keywords: repeat sample
2003-03-06 | GSE331 | GEO
Project description:Spleen total RNA (Catalog #64044-1) and liver total RNA (Catalog #64042-1) were purchased from Clontech and used as the starting material. Spleen was labeled with Cyanine-3 (Green) while liver was labeled with Cyanine-5 (Red). cRNA was made by the single round amplification method. Keywords: repeat sample
2003-03-06 | GSE330 | GEO
Project description:UMRR (Universal Mouse Reference RNA, Catalog #740100) was purchased from Stratagene and used as the starting material. Liver total RNA (Catalog #64042-1) was purchased from Clontech and used as the starting material. UMRR was labeled with Cyanine-3 (Green) while Liver was labeled with Cyanine-5 (Red). cRNA was made by the single round amplification method. Keywords: repeat sample
2003-03-07 | GSE333 | GEO
Project description:UMRR (Universal Mouse Reference RNA, Catalog #740100) was purchased from Stratagene and used as the starting material. Spleen total RNA (Catalog #64044-1) was purchased from Clontech and used as the starting material. UMRR was labeled with Cyanine-3 (Green) while spleen was labeled with Cyanine-5 (Red). cRNA was made by the single round amplification method. Keywords: repeat sample
2003-03-07 | GSE332 | GEO
Project description:Surgically resected gastric adenocarcinoma samples and their paired disease-free (R0) marginal nonmalignant tissue were obtained from 14 patients with gastric adenocarcinoma. RNA was isolated using the RNAeasy Kit (Qiagen Valencia, CA) from 15 mg of tissue. The quality of total RNA and its integrity was assessed using the Bioanalyzer 2100 (Agilent, Palo Alto, CA) and RIN value (RNA Integrity Number) was recorded for all the samples for intact 18S and 28S rRNA. Total RNA (800 ng) from each sample was reverse transcribed and linear amplified using the low RNA input linear amplification kit (Agilent Technologies). After synthesis of the first and second strand of cDNA, the product was used in an in vitro transcription reaction to generate cRNAs in the presence of cyanine 3-labeled UTP (Cy3) for normal and cyanine 5-labeled UTP (Cy5) for tumor. Fragmented Cy3-labeled cRNA of the control sample were mixed with equal amounts of Cy5-labeled cRNA from the gastric tumor sample, and the mixtures were hybridized onto 44K whole human genome DNA microarrays (Agilent Technologies) for 17 hrs at 65°C with constant rotation (10 rpm). Subsequently, the arrays were washed according to manufacturer’s instructions. The slides were scanned using an Agilent microarray scanner (G2565AA), and the images were processed using the Agilent feature extraction software AFE 9.5. GeneSpring GX v10.1 (Agilent Technologies) was used to analyze the expression profiles obtained after microarray hybridization. The feature extraction files from all the samples were uploaded to GeneSpring. Following intensity-dependent normalization (Lowess), the data was subjected to statistical analysis. A two tailed student’s t-test was used to determine significance of the differences observed between the normal and tumor samples. Benjamini Hochberg algorithm was used to compute false discovery rates. Genes were filtered based on P value threshold of 0.001 and false discovery rate of less than 1%.
2010-07-08 | GSE22804 | GEO
Project description:Gene expression analysis study in cerulenin treated - 7µg/ml cerulenin treated Y79 cells and control Y79 cells Organism used: Homo sapiens ï Slides: Gene Expression Whole Human Genome 4x44k ï Starting material: Cells in RNA later ï RNA Samples used: :Control Y79 and Treated_Cerulenin ï Labeling kit: Agilents Low input RNA linear amplification Kit one color ï Labeling Method: T7 promoter based-linear amplification to generate labeled complementary RNA ï Total RNA and cRNA Purification Kit: Qiagenâs RNeasy minikit ï Hybridization Kit: Agilentâs In situ Hybridzation kit Hybridization protocol The fragmented cRNA were mixed with 25ul of 2x GE Hybridization Buffer (Agilent). About 45ul of the resulting mixture was applied to the Microarray and hybridized at 65degC for 17 hours in an Agilent Microarray Hybridization Chamber (SureHyb: G2534A) with Hybridization Oven. After hybridization, slides were washed with Agilent Gene expression Wash Buffer I for 1 minute at room temperature followed by a 1 min wash with Agilent Gene expression Wash Buffer II for 37C. Slides were finally rinsed with Acetonitrile for cleaning up and drying. Scan protocol Laser detection of Cyanine 3 and Cyanine 5 fluorescence is performed using a confocal scanning instrument containing two tuned lasers, which excite Cyanine dyes at the appropriate wavelengths. Description Microarrays were scanned on an Agilent scanner (G2565AA) at 100% laser power, 30% PMT.Data extraction was carried out with Agilent Feature Extraction software (version 9.1), and normalization was done using linear per array algorithm according to the manufacturerâs protocol. Data processing Feature extracted data was analyzed using GeneSpring GX v 7.3.1 software from Agilent. Normalization of the data was done in GeneSpring GX using the recommended one color Per Chip and Per Gene Data Transformation: Set measurements less than 0.01 to 0.01, Per Chip: Normalize to 50th percentile, Per Gene: Normalize to Specific Samples. Further quality control of normalized data was done using correlation based condition tree to eliminate bad experiments. Differentially regulated Genes were filtered with cutoff of > 1.5 for Up regulation and < 0.55 for Down Regulation were obtained. Differentially regulated genes were clustered using gene tree to identify significant gene expression patterns.
2010-08-18 | E-GEOD-16999 | biostudies-arrayexpress
Project description:Gene expression analysis study in curcumin treated (20µM curcumin treated Y79 cells ) and control Y79 cells (Suspension Y79 cells). Source were Y79 retinoblastoma cell line from human. Organism used: Homo sapiens ï Slides: Gene Expression Whole Human Genome 4x44k ï Starting material: Cells in RNA later ï RNA Samples used: :Control Y79 and Treated_Curcumin ï Labeling kit: Agilents Low input RNA linear amplification Kit one color Labeling Method: T7 promoter based-linear amplification to generate labeled complementary RNA ï Total RNA and cRNA Purification Kit: Qiagenâs RNeasy minikit ï Hybridization Kit: Agilentâs In situ Hybridzation kit Hybridization protocol The fragmented cRNA were mixed with 25ul of 2x GE Hybridization Buffer (Agilent). About 45ul of the resulting mixture was applied to the Microarray and hybridized at 65degC for 17 hours in an Agilent Microarray Hybridization Chamber (SureHyb: G2534A) with Hybridization Oven. After hybridization, slides were washed with Agilent Gene expression Wash Buffer I for 1 minute at room temperature followed by a 1 min wash with Agilent Gene expression Wash Buffer II for 37C. Slides were finally rinsed with Acetonitrile for cleaning up and drying. Scan protocol Laser detection of Cyanine 3 and Cyanine 5 fluorescence is performed using a confocal scanning instrument containing two tuned lasers, which excite Cyanine dyes at the appropriate wavelengths. Description Microarrays were scanned on an Agilent scanner (G2565AA) at 100% laser power, 30% PMT.Data extraction was carried out with Agilent Feature Extraction software (version 9.1), and normalization was done using linear per array algorithm according to the manufacturerâs protocol. Data processing Feature extracted data was analyzed using GeneSpring GX v 7.3.1 software from Agilent. Normalization of the data was done in GeneSpring GX using the recommended one color Per Chip and Per Gene Data Transformation: Set measurements less than 0.01 to 0.01, Per Chip: Normalize to 50th percentile, Per Gene: Normalize to Specific Samples. Further quality control of normalized data was done using correlation based condition tree to eliminate bad experiments. Differentially regulated Genes were filtered with cutoff of > 1.5 for Up regulation and < 0.55 for Down Regulation were obtained. Differentially regulated genes were clustered using gene tree to identify significant gene expression patterns.
2010-08-18 | E-GEOD-17034 | biostudies-arrayexpress