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Modeling Formamide Denaturation of Probe-Target Hybrids for Improved Microarray Probe Design in Microbial Diagnostics

ABSTRACT: 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.

ORGANISM(S): Escherichia coli K-12 Cereibacter sphaeroides 2.4.1

PROVIDER: GSE33021 | GEO | 2012/06/30

SECONDARY ACCESSION(S): PRJNA148019

REPOSITORIES: GEO

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Modeling Formamide Denaturation of Probe-Target Hybrids for Improved Microarray Probe Design in Microbial Diagnostics

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

Modeling Formamide Denaturation of Probe-Target Hybrids for Improved Microarray Probe Design in Microbial Diagnostics

Project description:Modeling Formamide Denaturation of Probe-Target Hybrids for Improved Microarray Probe Design in Microbial Diagnostics

| PRJNA148019 | ENA

Temperature probe selection experiment for custom array production

Project description:Microarrays have evolved from low-density cDNA or oligonucleotide arrays to high-density platforms, for several study species even covering the complete transcriptome. At the same time, transcriptomics experiments have become more complex and multifactorial in nature, requiring many microarrays to assess multiple biologically relevant hypotheses. Scientists using this technology are therefore painfully aware of the high financial cost of a typical microarray experiment. Unfortunately, this often leads to either a suboptimal experimental design in an effort to reduce the cost by using fewer microarrays, or to abandoning microarray technology altogether. In this study, we argue that for many studies high-density full genome microarrays are in fact technical overkill. By selectively reducing full genome probe sets to a lower number of probes, it is possible to significantly reduce the total cost of a microarray experiment. The study consists of four microarray analyses: a cadmium probe selection experiment, a temperature probe selection experiment, a cadmium validation experiment and a cadmium validation experiment.

2009-12-31 | GSE17083 | GEO

Cadmium probe selection experiment for custom array production

2009-12-31 | GSE17082 | GEO

Microarray probe design

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

Genome-wide identification and analysis of microRNA expression in MEFs, ESCs, and iPSCs

Project description:Microarray analysis of miRNA—Two micrograms of total RNA was extended at the 3′ terminus with a poly(A) tail using poly(A) polymerase. An oligonucleotide tag was then ligated to the poly(A) tail for later fluorescent dye staining. Hybridization was performed overnight on a µParaflo microfluidic chip using a microcirculation pump (Atactic Technologies). On the microfluidic chip, each detection probe consisted of a chemically modified nucleotide coding segment complementary to the target microRNA (miRBase, http://mirbase.org) and a spacer segment of polyethylene glycol to extend the coding segment away from the substrate. The detection probes were made by in situ synthesis using photogenerated reagent chemistry. The hybridization melting temperatures were balanced by chemical modifications of the detection probes. Hybridization used 100 µl 6× SSPE buffer (0.90 M NaCl, 60 mM Na2HPO4, 6 mM EDTA, pH 6.8) containing 25% formamide at 34°C. After RNA hybridization, tag-conjugating Cy5 dye was circulated through the microfluidic chip for dye staining. Fluorescence images were collected by using a laser scanner (GenePix 4000B, Molecular Device) and digitized using Array-Pro image analysis software (Media Cybernetics). Data were analyzed by first subtracting the background and then normalizing the signals using a LOWESS filter (Locally-weighted Regression).

2015-10-01 | GSE65596 | GEO

Rice indica genome tiling experiments

Project description:This experiment was designed to identify transcribed regions of indica rice genome. A series of high-density oligonucleotide tiling arrays that represent sense and antisense strands of the entire nonrepetitive sequence of the chromosome were used to measure transcriptional activities. A total of 838,816 36mer oligonucleotide probes, positioned every 46 nt on average, were designed to interrogate the indica genome, respectively. The probes were synthesized via maskless photolithography at a feature density of approximately 389,000 probes per slide. The arrays were hybridized with fluorescence-labeled cDNA reverse-transcribed from equal amounts of four selected poly(A)+ RNA populations, namely, seedling roots, seedling shoots, panicles, and suspension cultured cells of the respective rice subspecies. Keywords: genome tiling experiments

2005-10-15 | GSE3452 | GEO

Tiling microarray of Oryza sativa japonica at 46nt resolution

Project description:This experiment was designed to identify transcribed regions of japonica subspecies of the rice genome. A series of high-density oligonucleotide tiling arrays that represent sense and antisense strands of the entire nonrepetitive sequence of all the 12 chromosomes were designed to measure genome-wide transcription. A total of 12253842 36mer oligonucleotide probes positioned every 46 nt on average were used for this purpose. The probes were synthesized via maskless photolithography at a feature density of approximately 389,000 probes per slide. The arrays were hybridized with fluorescence-labeled cDNA reverse-transcribed from equal amounts of four selected poly(A)+ RNA population (seedling root, seedling shoot, panicle, and suspension cultured cells). Keywords: tiling array, genome-wide transcription

2007-02-21 | GSE6996 | GEO

Homo sapiens

Project description:Improved detection of global copy number variation using high density, non-polymorphic oligonucleotide probes

| PRJNA102563 | ENA

Tiling microarray analysis of japonica and indica rice chromosome 10

Project description:This experiment was designed to identify transcribed regions of both japonica and indica rice chromosome 10. A series of high-density oligonucleotide tiling arrays that represent sense and antisense strands of the entire nonrepetitive sequence of the chromosome were used to measure transcriptional activities. A total of 750,282 and 838,816 36mer oligonucleotide probes, positioned every 46 nt on average, were designed to interrogating the japonica and the indica chromosome, respectively. The probes were synthesized via maskless photolithography at a feature density of approximately 389,000 probes per slide. The arrays were hybridized with fluorescence-labeled cDNA reverse-transcribed from equal amounts of four selected poly(A)+ RNA populations, namely, seedling roots, seedling shoots, panicles, and suspension cultured cells of the respective rice subspecies. Keywords: other

2005-04-09 | GSE2500 | GEO

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