DNA microarray analysis of CfMNPV genes
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ABSTRACT:
ORGANISM(S): Choristoneura fumiferana MNPV
SUBMITTER: Dan-Hui Yang
PROVIDER: E-GEOD-14747 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:Temporal expression profiles of all 146 ORFs, and their complements, of Choristoneura fumiferana nucleopolyhedrovirus (CfMNPV) were determined by a modified oligonucleotide based two-channel DNA microarray. Total RNA was isolated at different times post infection from Cf203 insect cells infected with CfMNPV. The cDNA was synthesized, fluorescently labeled with Cy3, and co-hybridized to the microarray chips along with Cy5-labeled CfMNPV viral genomic DNA (vgDNA), which was used as an equimolar reference standard for each gene. From the microarray data, the temporal gene expression profiles could readily be classified into four clusters based on timing of expression. Transcription of some non-coding antisense strands of the CfMNPV genes was also detected, which provided novel insights into viral gene functions.
2010-01-08 | GSE14747 | GEO
Project description:The temporal expression of the 23 CfMNPV genes representing all four temporal classes including its 7 unique genes were determined by a modified oligonucleotide-based two-channel DNA microarray. Transcription of the non-coding (antisense) strands of some of the CfMNPV select genes including the polyhedrin gene was also detected by the array analysis. The expression of four host genes varied several fold throughout virus infection. The microarray chip contained oligonucleotide probes for 23 CfMNPV ORFs and their complements. We first developed a novel normalization protocol using Cy5-labeled CfMNPV viral genomic DNA (vgDNA) as equimolar reference standards for each probe in order to overcome the inherent variability problem of the traditional microarray normalization procedures including use of internal standards. Cy3-labeled cDNA was from total RNA isolated at different times post infection of Cf203 insect cells infected with CfMNPV. Host genes were unsuitable for normalization between microarrays. The DNA microarray results were selectively validated by quantitative RT-PCR (qRT-PCR). The nature of the polyhedrin antisense transcription was further investigated using long range RT-PCR analysis. Keywords: Time course, detection of antisense transcripts, viral genomic DNA normalization Total RNA was isolated from Cf203 cells at 0, 3, 6, 12, 24 and 48 h post infection, as well as from mock-infected Cf203 cells. The Cy3-labeled cDNA derived from 20 μg total RNA was co-hybridized with 10 ng/μl vgDNA to each array at 42ºC. Seven hybridizations in each experiment, two independent hybridization experiments were performed, and therefore 14 samples were analyzed and included in the sample submission including the mock-infected sample.
2007-05-01 | E-GEOD-4783 | biostudies-arrayexpress
Project description:The temporal expression of the 23 CfMNPV genes representing all four temporal classes including its 7 unique genes were determined by a modified oligonucleotide-based two-channel DNA microarray. Transcription of the non-coding (antisense) strands of some of the CfMNPV select genes including the polyhedrin gene was also detected by the array analysis. The expression of four host genes varied several fold throughout virus infection. The microarray chip contained oligonucleotide probes for 23 CfMNPV ORFs and their complements. We first developed a novel normalization protocol using Cy5-labeled CfMNPV viral genomic DNA (vgDNA) as equimolar reference standards for each probe in order to overcome the inherent variability problem of the traditional microarray normalization procedures including use of internal standards. Cy3-labeled cDNA was from total RNA isolated at different times post infection of Cf203 insect cells infected with CfMNPV. Host genes were unsuitable for normalization between microarrays. The DNA microarray results were selectively validated by quantitative RT-PCR (qRT-PCR). The nature of the polyhedrin antisense transcription was further investigated using long range RT-PCR analysis. Keywords: Time course, detection of antisense transcripts, viral genomic DNA normalization
2007-05-01 | GSE4783 | GEO
Project description:The short length of miRNAs results in a high dynamic range of melting temperatures and therefore impedes a proper selection of detection probes or optimized PCR primers. While miRNA microarrays allow for massive parallel and accurate relative measurement of all known miRNAs, they have so far been less useful as an assay for absolute quantification. Here we developed a new method based not only to the hybridization process that presents the limits before described, but integrating the hybridization to an enzymatic reaction. Moreover we introduced spike-in in the hybridization-enzymatic reaction allowing the quantification of miRNAs respect to them, canceling biases related to sequence, labeling, or hybridization. An alternative method for the absolute miRNA quantization was recently proposed by Bissels (Absolute quantification of microRNAs by using a universal reference. RNA). It was based on the Absolute quantification of microRNAs by using a universal reference consisting of 954 synthetic human, mouse, rat, and viral miRNAs, with each individual oligoribonucleotide present in equimolar concentrations with tested miRNAs. Thereby, any single miRNA detected on a microarray can be quantified by directly comparing its signal intensity with the one obtained by the same miRNA sequence present in the universal reference adjusting for biases related to sequence, labeling, hybridization, or signal detection. Our method allowed the detection of a comparable concentration of miRNA (10^-18 moles to 10^-14 moles in a linear range), but allows controlling the hybridization quality and reproducibility basing on the results of the interpolation of the spike-in dependent curve. Moreover, our method does not influenced by phenomena imputable to different labeling process due to different sequences because labeling was due only to the incorporation of biotin-d(A) if the hybridized miRNA acted as primer for the klenow enzyme. This method allowed the discussion of miRNA genes expression in 9 different tissues relating them with tissue functionality in the cardiocirculatory system.
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2011-12-31 | E-GEOD-28142 | biostudies-arrayexpress
Project description:The short length of miRNAs results in a high dynamic range of melting temperatures and therefore impedes a proper selection of detection probes or optimized PCR primers. While miRNA microarrays allow for massive parallel and accurate relative measurement of all known miRNAs, they have so far been less useful as an assay for absolute quantification. Here we developed a new method based not only to the hybridization process that presents the limits before described, but integrating the hybridization to an enzymatic reaction. Moreover we introduced spike-in in the hybridization-enzymatic reaction allowing the quantification of miRNAs respect to them, canceling biases related to sequence, labeling, or hybridization. An alternative method for the absolute miRNA quantization was recently proposed by Bissels (Absolute quantification of microRNAs by using a universal reference. RNA). It was based on the Absolute quantification of microRNAs by using a universal reference consisting of 954 synthetic human, mouse, rat, and viral miRNAs, with each individual oligoribonucleotide present in equimolar concentrations with tested miRNAs. Thereby, any single miRNA detected on a microarray can be quantified by directly comparing its signal intensity with the one obtained by the same miRNA sequence present in the universal reference adjusting for biases related to sequence, labeling, hybridization, or signal detection. Our method allowed the detection of a comparable concentration of miRNA (10-18 moles to 10-14 moles in a linear range) (see Figure), but allows controlling the hybridization quality and reproducibility basing on the results of the interpolation of the spike-in dependent curve. Moreover, our method does not influenced by phenomena imputable to different labeling process due to different sequences because labeling was due only to the incorporation of biotin-d(A) if the hybridized miRNA acted as primer for the klenow enzyme. This method allowed the discussion of miRNA genes expression in 14 different tissues relating it with tissue anatomical proximity and functional similarity. 2K microarray was hybridized with small RNAs from 14 different tissues (Atrium Sinister; Skin; Liver; W_B_A: White Blood Cells from Arteriosum blood; W_B_V: White Blood Cells from Venosum blood; Lymph Node; Tongue; Spleen; Skeletal Muscle; Lung; Kidney; Stomach; Adipose Tissue; Ventricle Sinister). Each experiment was replicated to have 28 experiments and miRNA gene expression was correlated with mRNA gene expression in the same samples.
2014-03-31 | E-GEOD-28140 | biostudies-arrayexpress
Project description:To investigate the DNA binding specificity of the CLAMP protein, we have designed a custom PBM to interrogate the binding of CLAMP to DNA sequences extracted from the Drosophila melanogaster genome. Specific regions were extracted based on ChIP-seq data, motif occurence and proximity to gene transcription start sites. N-terminal GST-tagged protein samples were made for the C-terminal four and six zinc finger portions of the protein CLAMP; samples were made by in vitro transcription translation (IVT). IVT reaction mixtures for the two CLAMP constructs were applied directly to the PBM microarray and incubated for 1hour. Microarray-bound protein was fluorescently labeled using Alexa488-conjugated antibodies targeting GST, and the microarray was scanned in using a standard microarray scanner. Median fluorescence intensity over eight replicate probes was reported for each unique DNA sequence on the microarray.
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Project description:Rainbow smelt (Osmerus mordax) are freeze-resistant fish that accumulate glycerol and produce an antifreeze protein during winter. Quantitative reverse transcription-PCR (qPCR) and subtractive hybridization studies have revealed a few genes in smelt liver to be differentially regulated in winter in comparison with the fall when water temperatures are warmer. In order to further define the suite of processes that are regulated seasonally, we undertook a large-scale analysis of gene expression by hybridization of smelt cDNA to the salmonid 16K cGRASP cDNA microarray. These microarray experiments were conducted as a focused sieving exercise, which identified informative genes for further study in the microarray samples and over a seasonal sampling series using quantitative reverse-transcription PCR. Total RNA was obtained from livers of 3 male fish sampled on October 20th and aliquots were pooled to contain equimolar concentrations of each RNA preparation. Total RNA was prepared from 3 male fish on January 3rd and processed in the same manner. Fluorescently labeled cDNA preparations were made using the fall (October) and winter (January) pools and mixed before hybridization to the microarray. The cDNAs were hybridized to three separate microarrays in order to generate technical replicates. Differential expression was assessed in order to select genes for further study.
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Project description:A framework to validate fluorescently labeled DNA-binding proteins for single-molecule experiments
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Project description:Microarray based comparative genomic hybridization of mouse adapted strains: 10700 pre-mouse SS1, SS1(A.L.)Sydney strain obtained directly from Adrian Lee, NSH79 Salama lab strain labeled as SS1-now know distinct, G27 obtained from Antonello Covacci. Genomic DNA from est strain (Cy5), genomic DNA from reference strains used for array fabrication (equimolar mix of 26695 and J99 genomic DNA) An all pairs experiment design type is where all labeled extracts are compared to every other labeled extract. Keywords: all_pairs
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