Transcription profiling of Trypanosoma brucei BSF and PCF monomorphic cells
ABSTRACT: Cy3 and Cy5 direct labelled RNA from Bloodstream MiTat1.1 trypanosomes and Procyclic 427 Lister were hybridized onto JCVI Trypanosoma brucei oligoarrays (version2). Procyclic RNA were used as control for data analysis.
Project description:Cy3 and Cy5 direct labelled RNA from Bloodstream MiTat1.1 trypanosomes and Procyclic 427 Lister were hybridized onto JCVI Trypanosoma brucei oligoarrays (version2). Procyclic RNA were used as control for data analysis.
Project description:Comparison of gene expression profiles of wild-type and fl-mutant upland cotton ovules Data for six replicate experiments performed with biologically independent samples. Cy3 and Cy5 dyes were used as follows: Set 1: wt+5-Cy5 wt+3-Cy3 Set 1 (dye swap): wt+5-Cy3 wt+3-Cy5 Set 2: wt+5-Cy5 wt+3-Cy3 Set 2 (dye swap): wt+5-Cy3 wt+3-Cy5 Set 3: wt+5-Cy5 wt+3-Cy3 Set 3 (dye swap): wt+5-Cy3 wt+3-Cy5 GSM63341, GSM63342, GSM63343, GSM63344, GSM63345, GSM63346 Data for 3 replicate experiments performed with biologically independent samples. Cy3 and Cy5 dyes were used as follows: Set 1: wt+3-Cy5 vs wt+3-Cy3 Set 2: wt+3-Cy3 vs wt+3-Cy5 Set 3: wt+3-Cy5 vs wt+3-Cy3 GSM63365, GSM63366, GSM63367 Data for six replicate experiments performed with biologically independent samples. Cy3 and Cy5 dyes were used as follows: Set 1: wt+3-Cy5 wt-3-Cy3 Set 1 (dye swap): wt+3-Cy3 wt-3-Cy5 Set 2: wt+3-Cy5 wt-3-Cy3 Set 2 (dye swap): wt+3-Cy3 wt-3-Cy5 Set 3: wt+3-Cy5 wt-3-Cy3 Set 3 (dye swap): wt+3-Cy3 wt-3-Cy5 GSM63368, GSM63369, GSM63370, GSM63371, GSM63372, GSM63373 Data for six replicate experiments performed with biologically independent samples. Cy3 and Cy5 dyes were used as follows: Set 1: 0wt-Cy5 wt+3-Cy3 Set 1 (dye swap): 0wt-Cy3 wt+3-Cy5 Set 2: 0wt-Cy5 wt+3-Cy3 Set 2 (dye swap): 0wt-Cy3 wt+3-Cy5 Set 3: 0wt-Cy5 wt+3-Cy3 Set 3 (dye swap): 0wt-Cy3 wt+3-Cy5 GSM63374, GSM63375, GSM63376, GSM63377, GSM63378, GSM63379 Data for six replicate experiments performed with biologically independent samples. Cy3 and Cy5 dyes were used as follows: Set 1: wt+3-Cy5 fl+3-Cy3 Set 1 (dye swap): wt+3-Cy3 fl+3-Cy5 Set 2: wt+3-Cy5 fl+3-Cy3 Set 2 (dye swap): wt+3-Cy3 fl+3-Cy5 Set 3: wt+3-Cy5 fl+3-Cy3 Set 3 (dye swap): wt+3-Cy3 fl+3-Cy5 GSM63391, GSM63392, GSM63393, GSM63394, GSM63395, GSM63396 Data for six replicate experiments performed with biologically independent samples. Cy3 and Cy5 dyes were used as follows: Set 1: wt+10-Cy5 fl+10-Cy3 Set 1 (dye swap): wt+10-Cy3 fl+10-Cy5 Set 2: wt+10-Cy5 fl+10-Cy3 Set 2 (dye swap): wt+10-Cy3 fl+10-Cy5 Set 3: wt+10-Cy5 fl+10-Cy3 Set 3 (dye swap): wt+10-Cy3 fl+10-Cy5 GSM63397, GSM63398, GSM63399, GSM63400, GSM63401, GSM63402 Data for six replicate experiments performed with biologically independent samples. Cy3 and Cy5 dyes were used as follows: Set 1: wt+10-Cy5 wt+3-Cy3 Set 1 (dye swap): wt+10-Cy3 wt+3-Cy5 Set 2: wt+10-Cy5 wt+3-Cy3 Set 2 (dye swap): wt+10-Cy3 wt+3-Cy5 Set 3: wt+10-Cy5 wt+3-Cy3 Set 3 (dye swap): wt+10-Cy3 wt+3-Cy5 GSM63403, GSM63404, GSM63405, GSM63406, GSM63407, GSM63408 Data for six replicate experiments performed with biologically independent samples. Cy3 and Cy5 dyes were used as follows: Set 1: wt+15-Cy5 wt+3-Cy3 Set 1 (dye swap): wt+15-Cy3 wt+3-Cy5 Set 2: wt+15-Cy5 wt+3-Cy3 Set 2 (dye swap): wt+15-Cy3 wt+3-Cy5 Set 3: wt+15-Cy5 wt+3-Cy3 Set 3 (dye swap): wt+15-Cy3 wt+3-Cy5 GSM63409, GSM63410, GSM63411, GSM63412, GSM63413, GSM63414 Data for six replicate experiments performed with biologically independent samples. Cy3 and Cy5 dyes were used as follows: Set 1: wt+20-Cy5 wt+3-Cy3 Set 1 (dye swap): wt+20-Cy3 wt+3-Cy5 Set 2: wt+20-Cy5 wt+3-Cy3 Set 2 (dye swap): wt+20-Cy3 wt+3-Cy5 Set 3: wt+20-Cy5 wt+3-Cy3 Set 3 (dye swap): wt+20-Cy3 wt+3-Cy5 GSM63415, GSM63416, GSM63417, GSM63418, GSM63419, GSM63420
Project description:Covalent heterodimers of the Cy3 and Cy5 fluorophores have been prepared from commercially available starting materials and characterized at the single-molecule level. This system behaves as a discrete molecular photoswitch, in which photoexcitation of the Cy5 results in fluorescence emission or, with a much lower probability, causes the Cy5 to enter into a long-lived, but metastable, dark state. Photoinduced recovery of the emissive Cy5 is achieved by very low intensity excitation (5 W cm(-2)) of the Cy3 fluorophore at a shorter wavelength. A similar system consisting of proximal, but not covalently linked, Cy3 and Cy5 has found application in stochastic optical reconstruction microscopy (STORM), a single-molecule localization-based technique for super-resolution imaging that requires photoswitching. The covalent Cy3-Cy5 heterodimers described herein eliminate the need for probabilistic methods of situating the Cy3 and Cy5 in close proximity to enable photoswitching. As proof of principle, these heterodimers have been applied to super-resolution imaging of the tubular stalk structures of live Caulobacter crescentus bacterial cells.
Project description:Vascular Endothelial Growth Factor (VEGF) is a critical regulator of pulmonary Th2 inflammation but the underlying mechanism and the roles of miRNAs in this process have not been defined. We analyzed the effect of VEGF on lung microRNAs by microarray analysis and validated the findings by Taqman qRT-PCR. We compared the levels of microRNAs in the lungs of transgenic mice with their wild type litters after overexpression of VEGF from a lung epithelium-restricted transgene. VEGF was overexpressed in the lung epithelium of CC10-rtTA-VEGF transgenic mice by adding Doxycycline to their drinking water for 7-10 days. RNA from the lungs of VEGF transgenic mice and their wild type litters were extracted and analyzed by microarray. The expression levels of microRNAs that were significanly altered were validated in another group of mice. Lung RNA from each transgenic mouse was labeled with Cy5 and lung RNA from wild type mice were labeled with Cy3. The two labeled RNAs were hybridized to the miRNA array. This array (chip ID 01_M9.1_070362) detects miRNA transcripts listed in Sanger miRBase Release 9.1 (http://www.sanger.ac.uk/Software/Rfam/mirna/). Multiple control probes are included in each chip. The control probes are used for quality controls of chip production, sample labeling and assay conditions. Among the control probes, PUC2PM-20B and PUC2MM-20B are the perfect match and single-based match detection probes, respectively, of a 20-mer RNA positive control sequence that is spiked into the RNA samples before labeling. One may assess assay stringency from the intensity ratio of PUC2PM-20B and PUC2MM-20B, which is normally larger than 30. When the option for custom probes is selected, custom probes are also included. After hybridization three imaged were obtained. From Cy3 and Cy5 images one may directly read miRNA profiles and from Ratio images one may get a quick sense of differential expressions between the corresponding samples. The images are displayed in pseudo colors so as to expand visual dynamic range. In the Cy3 and Cy5 intensity images, as signal intensity increases from 1 to 65,535 the corresponding color changes from blue to green, to yellow, and to red. In the Cy3/Cy5 ratio image, when Cy3 level is higher than Cy5 level the color is green; when Cy3 level is equal to Cy5 level the color is yellow; and when Cy5 level is higher than Cy3 level the color is red.
Project description:Cy3 and Cy5 are among the most commonly used oligonucleotide labeling molecules. Studies of nucleic acid structure and dynamics use these dyes, and they are ubiquitous in microarray experiments. They are sensitive to their environment and have higher quantum yield when bound to DNA. The fluorescent intensity of terminal cyanine dyes is also known to be significantly dependent on the base sequence of the oligonucleotide. We have developed a very precise and high-throughput method to evaluate the sequence dependence of oligonucleotide labeling dyes using microarrays and have applied the method to Cy3 and Cy5. We used light-directed in-situ synthesis of terminally-labeled microarrays to determine the fluorescence intensity of each dye on all 1024 possible 5'-labeled 5-mers. Their intensity is sensitive to all five bases. Their fluorescence is higher with 5' guanines, and adenines in subsequent positions. Cytosine suppresses fluorescence. Intensity falls by half over the range of all 5-mers for Cy3, and two-thirds for Cy5. Labeling with 5'-biotin-streptavidin-Cy3/-Cy5 gives a completely different sequence dependence and greatly reduces fluorescence compared with direct terminal labeling.
Project description:To develop structural modifications of dNTPs that are compatible with Taq DNA polymerase activity, we synthesized eight dUTP derivatives conjugated with Cy3 or Cy5 dye analogues that differed in charge and charge distribution throughout the fluorophore. These dUTP derivatives and commercial Cy3- and Cy5-dUTP were studied in Taq polymerase-dependent polymerase chain reactions (PCRs) and in primer extension reactions using model templates containing one, two and three adjacent adenine nucleotides. The relative amounts of amplified DNA and the kinetic parameters Km and Vmax characterizing the incorporation of labelled dUMPs have been estimated using fluorescence measurements and analysed. The dUTPs labelled with electroneutral zwitterionic analogues of Cy3 or Cy5 fluorophores were used by Taq polymerase approximately one order of magnitude more effectively than the dUTPs labelled with negatively charged analogues of Cy3 or Cy5. The nucleotidyl transferase activity of Taq polymerase was also observed and resulted in the addition of dUMPs labelled with electroneutral or positively charged fluorophores to the 3' ends of DNA. The introduction of mutually compensating charges into fluorophores or other functional groups conjugated to dNTPs can be considered a basis for the creation of PCR-compatible modified nucleoside triphosphates.
Project description:Assays 1-4: Effects of DnaA activity on S. meliloti chromosome, pSymA and pSymB replicon bias. Sheared DNA extracts of IPTG-induced vs uninduced cultures of DnaA (Assays 1,2) or Hda (Assays 3,4) overexpressors were labeled with Cy3 or Cy5 and hybridized applying dye-swap replicate design. Assay 5: Hybridization of sheared DNA of logarithmic (Cy3-labeled) vs stationary phase (Cy5-labeled) culture.
Project description:Typical DNA microarrays utilize diffusion of dye-labeled cDNA probes followed by sequence-specific hybridization to immobilized targets. Here we experimentally estimated the distance typical probes travel during static 16-h hybridizations. Probes labeled with Cy3 and Cy5 were individually introduced to opposite sides of a microarray with minimal convective mixing. Oppositely labeled probes diffused across the initial front separating the two solutions, generating a zone with both dyes present. Diffusion-distance estimates for Cy3- and Cy5-labeled cDNAs were 3.8 mm and 2.6 mm, respectively, despite having almost identical molecular masses. In separate 16-h hybridization experiments with oppositely labeled probes premixed, arrays that were continuously mixed had 15-20% higher signal intensities than arrays hybridized statically. However, no change was observed in the Cy3/Cy5 signal intensity ratio between continuously mixed and static hybridizations. This suggests that the observed dye bias in diffusion-distance estimates results from differences in the detection limits of Cy3 and Cy5-labeled cDNA, a potential concern for array data on low-abundance transcripts. Our conservative diffusion-distance estimates indicate that replicate targets >7.6 mm apart will not compete for scarce probes. Also, raising the microarray gap height would delay the onset of diffusion-limited hybridization by increasing the amount of available probe.
Project description:S. pombe cells were challenged with 0.5 mM hydrogen peroxide for various periods of time (0, 10, 30, 60, 120, 180 minutes). Total RNA was isolated at each time point, labeled with Cy3 and hybridized on cDNA microarrays competitively with Cy5 labeled RNA pooled from all 6 samples. A biological replicate was performed in analyzed as a dyw swap (pool = Cy3, time series = Cy5).
Project description:Replication and segregation are the two main processes that maintain chromosomes in growing cells. In bacteria, replication and transcription have been proposed to provide motive force in chromosome segregation. Recently, ParB2, a segregation protein, encoded by V. cholerae chromosome II (chrII) was also found to influence chrII replication. V. cholerae chrII replication is primarily controlled by its specific initiator protein, RctB. Here, we have screened for ParB2 and RctB binding sites using a genome-wide DNA binding analysis (ChIP-chip). We report the identification of a new region containing additional RctB and ParB2 binding sites, and suggest the mechanisms to coordinate replication initiation with segregation of chromosomes. ParB2 binding DNA (Cy5) vs total DNA (input, Cy3) in wildtype (CVC209), RctB binding DNA (Cy5) vs total DNA (input, Cy3) in wildtype (CVC209), RctB binding DNA (Cy5) vs total DNA (input, Cy3) in MCH1 (CVC2099, rctB-deleted strain), Biological replicates: 3 or 2