Y. pestis exposed to various concentrations of ciprofloxacin for various time points (treated/nontreated analysis in each array).
Ontology highlight
ABSTRACT:
ORGANISM(S): Yersinia pestis
PROVIDER: GSE79687 | GEO | 2016/03/30
SECONDARY ACCESSION(S): PRJNA316706
REPOSITORIES: GEO
Similar Datasets
Project description:Cultures of Y. pestis Kimberley53 virulent strain were exposed to different concentrations of ciprofloxacin (including 0.016 µg/ml representing the strains' MIC value) for various time periods. Total RNA samples were extructed and used for cRNA synthesis and labelling (using Cy3-CTP for the treated samples and Cy5-CTP for the non-treated sample in one biological experiment and dye swap for the 2nd replicate of independent biological experiment). 8 Labeled cRNA samples were hybridized to custom made Agilent 8x15K slide (each slide represent 1 time point). Goal: Identifing alterations in gene expression profile which correlates with the ciprofloxacin induced growth inhibition. Those altered mRNA transcrips will be used as a markers for the development of rapid molecular Antibiotic Susceptibility Test. Y. perstis exposed to 0.001, 0.016, 0.5 and 4 µg/ml ciprofloxacin, compared to growth control (non treated) sample, for 3 time periods (45, 90 and 120 min.). Two independent biological replicates were performed. Extracted total RNA were used for cRNA synthesis and labeling with Cy3/Cy5-CTP, hybridized to Agilent custom-made 8x15K slide format.
2016-03-30 | E-GEOD-79687 | biostudies-arrayexpress
Project description:Cultures of Y. pestis Kimberley53 (Kim53) virulent strain and Kim53∆nlpD were grown in heart infusion broth supplemented with 0.2% xylose and 2.5 mM CaCl2 for 5 h at 37°C, 200rpm. Total RNA samples were extructed and used for cRNA synthesis and labelling (using Cy3-CTP and Cy5-CTP). cRNA of two independent samples from each strain were labeled and hybridized to custom made Agilent 8x15K slide. Goal: Identifing alterations in gene expression profile between the wild-type Kim53 virulent strain and Kim53∆nlpD. Those altered mRNA transcrips will be used for better understanding of the cause for Kim53∆nlpD attenuation.
2017-07-18 | GSE101490 | GEO
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.
2015-04-22 | GSE68094 | GEO
Project description:Rice is one of important crop and, the genome has been already completely sequenced. Moreover we collected more than 30K rice FL-cDNA clones as transcriptome resources. However, the cDNA collection did not reveal the transcription activity and specificity of expression. So, we designed 60-mer oligo array based on the array system of Agilent Technologies (GPL477) as custom array. For validation of the array, we tested the reproducibility of labeling and hybridization. After this validation, 22K rice oligo array was supplied from Agilent Technologies (GPL892). 1: reproducibility of labeling: mRNA of shoot sample was amplified and labeled more than 10 times with Cy3 and Cy5, and both Cy3- and Cy5-labeled cRNA was hybridized on a slide (self-hybridization), and validate the reproducibility of labeling. In addition, we also amplified and labeled mRNA of other samples with Cy3 and Cy5, and made a self-hybridization. The intensity data of these array was also indicated transcription activity of genes in these samples. Keywords: variety among tissues
2008-04-01 | GSE7530 | GEO
Project description:Sample labeling and array hybridization were performed according to the Agilent One-Color Microarray-Based Gene Expression Analysis protocol (Agilent Technology) with minor modifications. Briefly, mRNA was purified from total RNA after removal of rRNA (mRNA-ONLY™ Eukaryotic mRNA Isolation Kit, Epicentre). Then, each sample was amplified and transcribed into fluorescent cRNA along the entire length of the transcripts without 3' bias utilizing a random priming method (Arraystar Flash RNA Labeling Kit, Arraystar). The labeled cRNAs were purified by RNeasy Mini Kit (Qiagen). The concentration and specific activity of the labeled cRNAs (pmol Cy3/μg cRNA) were measured by NanoDrop ND-1000. 1 μg of each labeled cRNA was fragmented by adding 5 μl 10 × Blocking Agent and 1 μl of 25 × Fragmentation Buffer, then heated the mixture at 60°C for 30 min, finally 25 μl 2 × GE Hybridization buffer was added to dilute the labeled cRNA. 50 μl of hybridization solution was dispensed into the gasket slide and assembled to the LncRNA expression microarray slide. The slides were incubated for 17 hours at 65°C in an Agilent Hybridization Oven. The hybridized arrays were washed, fixed and scanned with using the Agilent DNA Microarray Scanner (part number G2505C).
2021-12-30 | GSE192662 | GEO
Project description:Rice Transcriptome - Slide A; Slide B
| PRJNA92201 | ENA
Project description:ratio experiment by combining dye-reversal ratio profiles CD4+ alpha beta T cells were positively sorted from LatY136F or from wild-type spleen using a MACS and anti-CD4 beads. Gamma delta T cells were sorted from the spleen of Lat3YF and of Eb-/- mice using MACS and anti-CD5 beads. In vitro differentiated Th1 and Th2 cells were generated by culturing naive wild-type CD4+ T cells for 4 days in complete RPMI in the presence of anti-CD3 (1 µg/ml) plus anti-CD28 (1 µg/ml) with the addition of IL-12 (10ng/ml) and of anti-IL-4 (5µg/ml) for Th1-polarizing conditions, and of IL-4 (20ng/ml) plus anti-IFN-gamma (10µg/ml) for Th2-polarizing conditions. RNA was prepared using the Trizol method followed by DNAse digestion. Microarray experiments were carried out as two-color ratio hybridizations. RNA labeling was performed with a Fluorescent Linear Amplification Kit (Agilent Technologies). In brief, cDNA was reverse transcribed from 4 µg total RNA with an oligo-dT-T7 promoter primer and MMLV-RT. Second strand synthesis was carried out with random hexamers. Fluorescent antisense cRNA was synthesized with either cyanine 3-CTP (Cy3-CTP) or cyanine 5-CTP (Cy5-CTP) and T7 polymerase. The fluorescent-labeled antisense cRNA was precipitated over night with LiCl, ethanol washed and resuspended in water. The purified products were quantified at A552nm for Cy3-CTP and A650nm for Cy5-CTP and labeling efficiency was verified with a Nanodrop photometer (Kisker, Steinfurt, Germany). Before hybridization, 1.25 µg labeled cRNA of each product were fragmented and mixed with control targets and hybridization buffer according to the supplier's protocol (Agilent Technologies). Hybridizations were done over night for approximately 17 h at 60°C. The slides were washed according to the manufacturer's manual and scanning of microarrays was performed with 5 µm resolution using a DNA microarray laser scanner (Agilent Technologies). In order to compensate dye specific effects, and to ensure statistically relevant data, a color swap dye reversal was performed. Features were extracted with an image analysis tool Version A 6.1.1 (Agilent Technologies) using default settings. Data analysis was carried out on the Rosetta Inpharmatics platform Resolver Built 4.0. Expression patterns were identified by stringent data analysis using anti-correlation of the dye reversal ratio profiles and a 2fold expression cut-off of the ratio experiments. By combining the first and the second criteria of analysis we filtered out data points with low P-value (P-value < 0.01), making the analysis robust and reproducible. Additionally, by using this strategy we did the data selection independent of error models implemented in the Rosetta Resolver system. Keywords: other
2005-03-01 | GSE2287 | GEO
Project description:ratio experiment by combining dye-reversal ratio profiles CD4+ alpha beta T cells were positively sorted from LatY136F or from wild-type spleen using a MACS and anti-CD4 beads. Gamma delta T cells were sorted from the spleen of Lat3YF and of Eb-/- mice using MACS and anti-CD5 beads. In vitro differentiated Th1 and Th2 cells were generated by culturing naive wild-type CD4+ T cells for 4 days in complete RPMI in the presence of anti-CD3 (1 µg/ml) plus anti-CD28 (1 µg/ml) with the addition of IL-12 (10ng/ml) and of anti-IL-4 (5µg/ml) for Th1-polarizing conditions, and of IL-4 (20ng/ml) plus anti-IFN-gamma (10µg/ml) for Th2-polarizing conditions. RNA was prepared using the Trizol method followed by DNAse digestion. Microarray experiments were carried out as two-color ratio hybridizations. RNA labeling was performed with a Fluorescent Linear Amplification Kit (Agilent Technologies). In brief, cDNA was reverse transcribed from 4 µg total RNA with an oligo-dT-T7 promoter primer and MMLV-RT. Second strand synthesis was carried out with random hexamers. Fluorescent antisense cRNA was synthesized with either cyanine 3-CTP (Cy3-CTP) or cyanine 5-CTP (Cy5-CTP) and T7 polymerase. The fluorescent-labeled antisense cRNA was precipitated over night with LiCl, ethanol washed and resuspended in water. The purified products were quantified at A552nm for Cy3-CTP and A650nm for Cy5-CTP and labeling efficiency was verified with a Nanodrop photometer (Kisker, Steinfurt, Germany). Before hybridization, 1.25 µg labeled cRNA of each product were fragmented and mixed with control targets and hybridization buffer according to the supplier's protocol (Agilent Technologies). Hybridizations were done over night for approximately 17 h at 60°C. The slides were washed according to the manufacturer's manual and scanning of microarrays was performed with 5 µm resolution using a DNA microarray laser scanner (Agilent Technologies). In order to compensate dye specific effects, and to ensure statistically relevant data, a color swap dye reversal was performed. Features were extracted with an image analysis tool Version A 6.1.1 (Agilent Technologies) using default settings. Data analysis was carried out on the Rosetta Inpharmatics platform Resolver Built 4.0. Expression patterns were identified by stringent data analysis using anti-correlation of the dye reversal ratio profiles and a 2fold expression cut-off of the ratio experiments. By combining the first and the second criteria of analysis we filtered out data points with low P-value (P-value < 0.01), making the analysis robust and reproducible. Additionally, by using this strategy we did the data selection independent of error models implemented in the Rosetta Resolver system. Keywords: other
2005-03-01 | GSE2286 | GEO
Project description:Total RNA was quantified by the NanoDrop ND-2000 (Thermo Scientific)and the RNA integrity was assessed using Agilent Bioanalyzer 2100 (Agilent Technologies). The sample labeling, microarray hybridization and washing were performed based on the manufacturer's standard protocols. Briefly, total RNA were transcribed to double strand cDNA, then synthesized into cRNA and labeled with Cyanine-3-CTP. The labeled cRNAs were hybridized onto the microarray. After washing, the arrays were scanned by the Agilent Scanner G2505C (Agilent Technologies).
2021-02-04 | GSE166057 | GEO
Project description:Changes of genome-wide mRNA transcription levels of human ciliary smooth muscle (hCSM) cells were determined by treating hCSM cells in culture with 200 nM of either an prostaglandin E2 receptor subtype EP2 or subtype EP4 selective agonist for 6 hours in comparison to untreated controls. This was followed by competitive hybridization of fluorescent Cy3 or Cy5 labeled cRNA probes derived from the treated versus untreated control total RNA samples onto an Agilent Human Whole Genome Expression oligonucleotide microarray. Log 2 (LN) of the intra-slide ratios (RATIO, PRE_VALUE) of treated versus untreated samples was reported as VALUE in the sample files. Keywords: prostaglandin E2 receptor agonists, subtype EP2, subtype EP4, hCSM
2008-02-24 | GSE10621 | GEO