Modeling Formamide Denaturation of Probe-Target Hybrids for Improved Microarray Probe Design in Microbial Diagnostics
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PROVIDER: PRJNA148019 | ENA |
REPOSITORIES: ENA
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Project description:This study aimed to model formamide-based melting for the optimization of the sensitivity and specifcity of oligonucleotide probes in dignostic high-density microarrays. Formamide melting profiles of DNA oligonucleotides were obtained with a high-density microarray targeting 16S rRNA genes of Escherichia coli and Rhodobacter sphaeroides. One or two mismatched versions of perfect match probes were included on the array to systematically analyze the effect of formamide on mismatch stability and mismatch discrimination. A thermodynamics-based mathematical model of formamide denaturation was developed to predict the formamide melting profiles with sufficient accuracy to help with oligonucleotide design in microbial ecology applications. 16S rRNA sequences with GenBank accession codes U00006 ( E. coli ) and X53853 (R. sphaeroides) were used for probe design. The following oligonucleotide probe sets were used for the systematic analysis of the effect of formamide on probe-target hybrids (parenthetic information gives set name followed by the number of probes): 22-mer perfect match probes tiling the 16S rRNA gene of E. coli (TileE, n=1521), perfect match E.coli probes of variable length between 18 and 26 mers (Length, n=1045), E. coli probes with central single mismatches (OneM, n=1563), E. coli probes with single positional mismatches (PosM, n=4092), E. coli probes with single deletion mismatches (Gap, n=248), E. coli probes with single insertion mismatches (Insertion, n=248), E. coli probes with two separate mismatches (TwoM, n=1674), E. coli probes with central tandem mismatches (Tandem, n=558), and 22-mer perfect match probes tiling the 16S rRNA gene of R. sphaeroides. Also, a probe with no match to 16S rRNA genes was used as a background control. On the array, regular probes were replicated three times and the Nonsense probe ten times. See the manuscript of Yilmaz et al. for details.
2012-06-29 | E-GEOD-33021 | biostudies-arrayexpress
Project description:This study aimed to model formamide-based melting for the optimization of the sensitivity and specifcity of oligonucleotide probes in dignostic high-density microarrays. Formamide melting profiles of DNA oligonucleotides were obtained with a high-density microarray targeting 16S rRNA genes of Escherichia coli and Rhodobacter sphaeroides. One or two mismatched versions of perfect match probes were included on the array to systematically analyze the effect of formamide on mismatch stability and mismatch discrimination. A thermodynamics-based mathematical model of formamide denaturation was developed to predict the formamide melting profiles with sufficient accuracy to help with oligonucleotide design in microbial ecology applications.
2012-06-30 | GSE33021 | GEO
Project description: Not available
| S-EPMC3428302 | biostudies-literature
Project description:MicroRNAs (miRNAs) have been shown to play an important role in many different cellular, developmental, and physiological processes. Accordingly, numerous methods have been established to identify and quantify miRNAs. The shortness of miRNA sequence results in a high dynamic range of melting temperatures and, moreover, 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 present a microarray based approach for global and absolute quantification of miRNAs. The method relies on an equimolar pool of about 1000 synthetic miRNAs of known concentration which is used as an universal reference and labeled and hybridized in a dual colour approach on the same array as the sample of interest. Each single miRNA is quantified with respect to the universal reference outbalancing bias related to sequence, labeling, hybridization or signal detection method. We demonstrate the accuracy of the method by various spike in experiments. Further, we quantified miRNA copy numbers in liver samples and CD34(+)CD133(-) hematopoietic stem cells. Different probe designs were investigated with respect to sensitivity and selectivity of microarray hybridization. An array with 11 different variants of oligonucleotides for miR-122a and miR-16 was produced. 5 µg of respective total RNA was fluorescently labelled by 3’ ligation. Total RNA was hybridized in a dual colour approach to microarrays versus a second labelled synthetic miRNA pool. The synthetic miRNA pool consisted of 5 fmol of each of 816 non redundant miRNAs sequences and miRControl 3 sequences. Local background was subtracted from the signal to obtain the net signal intensity and the mean of the net signal intensities of 4 corresponding spots representing the same miRNA was computed for those spots only which were unflagged (empty spots, poor spots, negative spots) and for which the fluorescent intensity of the miRNAs derived from the samples of interest was two-fold the mean background value (2bkg dataset).
2009-10-26 | E-GEOD-15832 | biostudies-arrayexpress
Project description:HiSpOD is a new efficient functional microarrays probe design algorithm especially dedicated for the microbial ecology and environmental studies. It was used to design 3392 probes targeting 21 genes involved in chlorinated solvent biodegradation pathways and synthesized on a nimblegen microarray. In order to test the probe specificity, the microarray was firstly hybridized to 6 µg of labelled aRNA from sheep rumen content (background aRNA). Secondly, hybridization of 1011 copies of labelled aRNA derived from in vitro transcription of three synthetic genes (mmoC, vcrA and tceA) and mixed with 6 µg of the same complex background material were performed to test their sensibility. Finally, the expression analysis of a contaminated groundwater sample was performed.
2011-04-13 | GSE21492 | GEO
Project description:microRNA-seq in circCDYL probe and NC probe
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Project description:MicroRNAs (miRNAs) have been shown to play an important role in many different cellular, developmental, and physiological processes. Accordingly, numerous methods have been established to identify and quantify miRNAs. The shortness of miRNA sequence results in a high dynamic range of melting temperatures and, moreover, 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 present a microarray based approach for global and absolute quantification of miRNAs. The method relies on an equimolar pool of about 1000 synthetic miRNAs of known concentration which is used as an universal reference and labeled and hybridized in a dual colour approach on the same array as the sample of interest. Each single miRNA is quantified with respect to the universal reference outbalancing bias related to sequence, labeling, hybridization or signal detection method. We demonstrate the accuracy of the method by various spike in experiments. Further, we quantified miRNA copy numbers in liver samples and CD34(+)CD133(-) hematopoietic stem cells.
2009-10-27 | GSE15832 | GEO
Project description:Oncolytic viruses are complex biological agents that interact at multiple levels with both tumor and normal tissues. Anti-viral pathways induced by interferon are known to play a critical role in determining tumor cell sensitivity and normal cell resistance to infection with oncolytic viruses. Here we pursue a synthetic biology approach to identify methods that enhance anti-tumor activity of oncolytic viruses through suppression of IFN signaling. Based on the mathematical analysis of multiple strategies, we hypothesize that a positive feedback loop, established by virus-mediated expression of a soluble interferon-binding decoy receptor, increases tumor cytotoxicity without compromising normal cells. Oncolytic rhabodviruses engineered to express a secreted interferon antagonist have improved oncolytic potential in cellular cancer models, and display improved therapeutic potential in tumor-bearing mice. Our results demonstrate the potential of this methodology in evaluating potential caveats of viral immune evasion strategies and improving the design of oncolytic viruses. The following series of microarray experiments was utilized to assess the impact of cloning an IFN decoy receptor isolated from vaccinia virus termed B19R on the transcriptional response against an IFN sensitive maraba virus strain termed MG1. RNA extraction was performed 24h post infection in 786-0 cells. Duplicate samples were pooled, and hybridized on Affymetrix human gene 1.0 ST arrays according to manufacturer instructions. Data analysis was performed using AltAnalyze. Briefly, probeset filtering implemented a DABG threshold of 70 & pV<0.05 and utilized exclusively constitutively expressed exons to assess levels of gene expression.
2013-06-01 | E-MTAB-1636 | biostudies-arrayexpress
Project description:The objective of this work was to design an improved host platform for recombinant protein expression in E. coli. The approach involves first to create a library of the E. coli genomic DNA in different expression vectors and screen for probable transcripts which may lead to slow growing colonies and also simultaneously over-expression of recombinant proteins. To observe its effect on host performance, these genes were knocked out from the E. coli genome. A CG2 strain has been created by knocking in vhb gene gene downstream of the acetate promoter and knocking down ribB gene in DH5α and transformed with Recombinant GFP cloned in pBAD33.
2011-05-24 | GSE29440 | GEO