The isotope array, a new tool that employs substrate-mediated labeling of rRNA for determination of microbial community structure and function

A new microarray method, the isotope array approach, for identifying microorganisms which consume a (14)C-labeled substrate within complex microbial communities was developed. Experiments were performed with a small microarray consisting of oligonucleotide probes targeting the 16S rRNA of ammonia-oxidizing bacteria (AOB). Total RNA was extracted from a pure culture of Nitrosomonas eutropha grown in the presence of [(14)C]bicarbonate. After fluorescence labeling of the RNA and microarray hybridization, scanning of all probe spots for fluorescence and radioactivity revealed that specific signals were obtained and that the incorporation of (14)C into rRNA could be detected unambiguously. Subsequently, we were able to demonstrate the suitability of the isotope array approach for monitoring community composition and CO(2) fixation activity of AOB in two nitrifying activated-sludge samples which were incubated with [(14)C]bicarbonate for up to 26 h. AOB community structure in the activated-sludge samples, as predicted by the microarray hybridization pattern, was confirmed by quantitative fluorescence in situ hybridization (FISH) and comparative amoA sequence analyses. CO(2) fixation activities of the AOB populations within the complex activated-sludge communities were detectable on the microarray by (14)C incorporation and were confirmed independently by combining FISH and microautoradiography. AOB rRNA from activated sludge incubated with radioactive bicarbonate in the presence of allylthiourea as an inhibitor of AOB activity showed no incorporation of (14)C and thus was not detectable on the radioactivity scans of the microarray. These results suggest that the isotope array can be used in a PCR-independent manner to exploit the high parallelism and discriminatory power of microarrays for the direct identification of microorganisms which consume a specific substrate in the environment.     

Application and validation of DNA microarrays for the 16S rRNA-based analysis of marine bacterioplankton

An oligonucleotide probe-based DNA microarray was evaluated for its ability to detect 16S rRNA targets in marine bacterioplankton samples without prior amplification by polymerase chain reaction (PCR). The results obtained were compared with those of quantitative fluorescence in situ hybridization (FISH). For extraction and direct labelling of total RNA, a fast and efficient protocol based on commercially available kits was established. A set of redundant and hierarchically structured probes was applied, and specificity of hybridization was assessed by additional control oligonucleotides comprising single central mismatches. The protocol was initially tested by microarray analysis of bacterial pure cultures. Complete discrimination of all control oligonucleotides was achieved, indicating a high degree of hybridization specificity. In a co-culture, abundant members were detected by microarray analysis, but signal ratios of positive probes did not correlate well with quantitative data from FISH experiments. A marine picoplankton sample from the German Bight was analysed. Bacterial populations with relative abundances of at least 5% were detected by hybridizing 0.1 microg of total RNA extracted from a sample of 375 ml equivalent to 4.1 x 10(8) cells. Our results demonstrate that major populations of marine bacterioplankton can be identified by microarray analysis in a fast and reliable way, even in relatively low volumes of sea water.     

The Isotope Array, a New Tool That Employs Substrate-Mediated Labeling of rRNA for Determination of Microbial Community Structure and Function

A new microarray method, the isotope array approach, for identifying microorganisms which consume a ¹⁴C-labeled substrate within complex microbial communities was developed. Experiments were performed with a small microarray consisting of oligonucleotide probes targeting the 16S rRNA of ammonia-oxidizing bacteria (AOB). Total RNA was extracted from a pure culture of Nitrosomonas eutropha grown in the presence of [¹⁴C]bicarbonate. After fluorescence labeling of the RNA and microarray hybridization, scanning of all probe spots for fluorescence and radioactivity revealed that specific signals were obtained and that the incorporation of ¹⁴C into rRNA could be detected unambiguously. Subsequently, we were able to demonstrate the suitability of the isotope array approach for monitoring community composition and CO₂ fixation activity of AOB in two nitrifying activated-sludge samples which were incubated with [¹⁴C]bicarbonate for up to 26 h. AOB community structure in the activated-sludge samples, as predicted by the microarray hybridization pattern, was confirmed by quantitative fluorescence in situ hybridization (FISH) and comparative amoA sequence analyses. CO₂ fixation activities of the AOB populations within the complex activated-sludge communities were detectable on the microarray by ¹⁴C incorporation and were confirmed independently by combining FISH and microautoradiography. AOB rRNA from activated sludge incubated with radioactive bicarbonate in the presence of allylthiourea as an inhibitor of AOB activity showed no incorporation of ¹⁴C and thus was not detectable on the radioactivity scans of the microarray. These results suggest that the isotope array can be used in a PCR-independent manner to exploit the high parallelism and discriminatory power of microarrays for the direct identification of microorganisms which consume a specific substrate in the environment.     

Circ_0136474 and MMP‐13 suppressed cell proliferation by competitive binding to miR‐127‐5p in osteoarthritis

Osteoarthritis (OA) is a prevalent degenerative joint disease whose pathogenesis remains unclear. The research aims to investigate the roles of Circ_0136474/miR‐127‐5p/MMP‐13 axis in OA. Differentially expressed circRNAs and miRNAs in OA cartilage tissue were screened out and visualized by R project based on RNA‐seq data and microarray data respectively. qRT‐PCR was carried out for detection of relative expression levels of Circ_0136474, miR‐127‐5p, MMP‐13 and other inflammatory factors and Western blot analysis was conducted to detect the protein expression level of MMP‐13. CCK‐8 assay and flow cytometry were conducted to determine cell proliferation and cell apoptotic ability respectively. RNA‐fluorescence in situ hybridization (RNA‐FISH) experiments were conducted to confirm the immune‐localization of the Circ_0136474 and MMP‐13 in human tissues. Targeted relationships were predicted by bioinformatic analysis and verified by dual‐luciferase reporter assay. Our findings revealed that the expression levels of both Circ_0136474 and MMP‐13 in OA cartilage tissue were significantly higher than that in normal cartilage tissue. Circ_0136474 could suppress cell proliferation by facilitating MMP‐13 expression and suppressing miR‐127‐5p expression in OA. Overexpression of miR‐127‐5p negatively regulated MMP‐13 expression to enhance cell proliferation. Our study demonstrated that Circ_0136474 and MMP‐13 suppressed cell proliferation, while enhanced cell apoptosis by competitive binding to miR‐127‐5p in OA, which may well provide us with a new therapeutic strategy for osteoarthritis.     

Combined DNA-RNA Fluorescent In situ Hybridization (FISH) to Study X Chromosome Inactivation in Differentiated Female Mouse Embryonic Stem Cells

Fluorescent in situ hybridization (FISH) is a molecular technique which enables the detection of nucleic acids in cells. DNA FISH is often used in cytogenetics and cancer diagnostics, and can detect aberrations of the genome, which often has important clinical implications. RNA FISH can be used to detect RNA molecules in cells and has provided important insights in regulation of gene expression. Combining DNA and RNA FISH within the same cell is technically challenging, as conditions suitable for DNA FISH might be too harsh for fragile, single stranded RNA molecules. We here present an easily applicable protocol which enables the combined, simultaneous detection of Xist RNA and DNA encoded by the X chromosomes. This combined DNA-RNA FISH protocol can likely be applied to other systems where both RNA and DNA need to be detected.     

An oligonucleotide prokaryotic acidophile microarray: its validation and its use to monitor seasonal variations in extreme acidic environments with total environmental RNA

An oligonucleotide microarray that monitors prokaryotic diversity in extremely acidic environments has been developed. The oligonucleotide probes target most known acidophilic microorganisms, including members of the Nitrospira phylum, Acidithiobacillus genus, acidobacteria, sulfur reducing bacteria, Actinobacteria and Archaea of the Ferroplasma and Thermoplasma genera. The probes were tested for their specificity against the corresponding type strain by microarray hybridization using PCR-amplified fluorescent DNA of the 16S rRNA genes. The microarray was tested and validated against well-established molecular ecology techniques such as molecular cloning and sequencing and FISH by using samples obtained from a natural extremely acidic environment, the Río Tinto (SW Spain). Also, fluorescent labelled total environmental RNA from Río Tinto samples were used as targets for microarray hybridizations. This approach allowed the detection of the most metabolically active prokaryotes of the ecosystem by simultaneously checking probes against 16S and 23S rRNAs as well as other functional genes. Seasonal and spatial variations in the relative expression of specific rRNA genes have been detected between two sampling sites that differ in several physicochemical parameters, mainly iron and sulfur content.     

Genomic profiling using array comparative genomic hybridization define distinct subtypes of diffuse large b-cell lymphoma: a review of the literature

ABSTRACT: Diffuse large B-cell lymphoma (DLBCL) is the most common type of non-Hodgkin Lymphoma comprising of greater than 30 % of adult non-Hodgkin Lymphomas. DLBCL represents a diverse set of lymphomas, defined as diffuse proliferation of large B lymphoid cells. Numerous cytogenetic studies including karyotypes and fluorescent in situ hybridization (FISH), as well as morphological, biological, clinical, microarray and sequencing technologies have attempted to categorize DLBCL into morphological variants, molecular and immunophenotypic subgroups, as well as distinct disease entities. Despite such efforts, most lymphoma remains undistinguishable and falls into DLBCL, not otherwise specified (DLBCL-NOS). The advent of microarray-based studies (chromosome, RNA, gene expression, etc) has provided a plethora of high-resolution data that could potentially facilitate the finer classification of DLBCL. This review covers the microarray data currently published for DLBCL. We will focus on these types of data; 1) array based CGH; 2) classical CGH; and 3) gene expression profiling studies. The aims of this review were three-fold: (1) to catalog chromosome loci that are present in at least 20 % or more of distinct DLBCL subtypes; a detailed list of gains and losses for different subtypes was generated in a table form to illustrate specific chromosome loci affected in selected subtypes; (2) to determine common and distinct copy number alterations among the different subtypes and based on this information, characteristic and similar chromosome loci for the different subtypes were depicted in two separate chromosome ideograms; and, (3) to list re-classified subtypes and those that remained indistinguishable after review of the microarray data. To the best of our knowledge, this is the first effort to compile and review available literatures on microarray analysis data and their practical utility in classifying DLBCL subtypes. Although conventional cytogenetic methods such as Karyotypes and FISH have played a major role in classification schemes of lymphomas, better classification models are clearly needed to further understanding the biology, disease outcome and therapeutic management of DLBCL. In summary, microarray data reviewed here can provide better subtype specific classifications models for DLBCL.     

Molecular cytogenetics in haematological malignancy: current technology and future prospects

Cytogenetics has played a pivotal role in haematological malignancy, both as an aid to diagnosis and in identifying recurrent chromosomal rearrangements, an essential prerequisite to identifying genes involved in leukaemia and lymphoma pathogenesis. In the late 1980s, a series of technologies based around fluorescence in situ hybridisation (FISH) revolutionised the field. Interphase FISH, multiplex-FISH (M-FISH, SKY) and comparative genomic hybridisation (CGH) have emerged as the most significant of these. More recently, microarray technologies have come to prominence. In the acute leukaemias, the finding of characteristic gene expression signatures corresponding to biological subgroups has heralded gene expression profiling as a possible future alternative to current cytogenetic and morphological methods for diagnosis. In the lymphomas, high-resolution array CGH has successfully identified new regions of deletion and amplification, providing the prospect of disease-specific arrays. Review related to the 15th International Chromosome Conference (ICC XV), held in September 2004, Brunel University, London, UK     

Evaluating gene expression dynamics using pairwise RNA FISH data

Recently, a novel approach has been developed to study gene expression in single cells with high time resolution using RNA Fluorescent In Situ Hybridization (FISH). The technique allows individual mRNAs to be counted with high accuracy in wild-type cells, but requires cells to be fixed; thus, each cell provides only a "snapshot" of gene expression. Here we show how and when RNA FISH data on pairs of genes can be used to reconstruct real-time dynamics from a collection of such snapshots. Using maximum-likelihood parameter estimation on synthetically generated, noisy FISH data, we show that dynamical programs of gene expression, such as cycles (e.g., the cell cycle) or switches between discrete states, can be accurately reconstructed. In the limit that mRNAs are produced in short-lived bursts, binary thresholding of the FISH data provides a robust way of reconstructing dynamics. In this regime, prior knowledge of the type of dynamics--cycle versus switch--is generally required and additional constraints, e.g., from triplet FISH measurements, may also be needed to fully constrain all parameters. As a demonstration, we apply the thresholding method to RNA FISH data obtained from single, unsynchronized cells of Saccharomyces cerevisiae. Our results support the existence of metabolic cycles and provide an estimate of global gene-expression noise. The approach to FISH data presented here can be applied in general to reconstruct dynamics from snapshots of pairs of correlated quantities including, for example, protein concentrations obtained from immunofluorescence assays.     

Modeling nonlinearity in dilution design microarray data

MOTIVATION: Dilution design (Mixed tissue RNA) has been utilized by some researchers to evaluate and assess the performance of multiple microarray platforms. Current microarray data analysis approaches assume that the quantified signal intensities are linearly related to the expression of the corresponding genes in the sample. However, there are sources of nonlinearity in microarray expression measurements. Such nonlinearity study in the expressions of the RNA mixtures provides a new way to analyze gene expression data, and we argue that the nonlinearity can reveal novel information for microarray data analysis. Therefore, we proposed a statistical model, called proportion model, which is based on the linear regression analysis. To approximately quantify the nonlinearity in the dilution design, a new calibration, beta ratio (BR) was derived from the proportion model. Furthermore, a new adjusted fold change (adj-FC) was proposed to predict the true FC without nonlinearity, in particular for large FC. RESULTS: We applied our method to one microarray dilution dataset. The experimental results indicated that, to some extent, there are global biases comparing with the linear assumption for the significant genes. Further analysis of those highly expressed genes with significant nonlinearity revealed some promising results, e.g. 'poison' effect was discovered for some genes in RNA mixtures. The adj-FCs of those genes with 'poison' effect, indicate that the nonlinearity can be also caused by the inherent feature of the genes besides signal noise and technical variation. Moreover, when percentage of overlapping genes (POG) was used as a cross-platform consistency measure, adj-FC outperformed simple fold change to show that Affymetrix and Illumina platforms are consistent. AVAILABILITY: The R codes which implements all described methods, and some Supplementary material, are freely available from http://www.utdallas.edu/~ying.liu/BetaRatio.htm     

Ultrasensitive DNA chip: gene expression profile analysis without RNA amplification

We have developed a new DNA chip whose substrate has a unique minute columnar array structure made of plastic. The DNA chip exhibits ultrahigh sensitivity, up to 100-fold higher than that of reference DNA chips, which makes it possible to monitor gene expression profiles even with very small amounts of RNA (0.1-0.01 microg of total RNA) without amplification. Differential expression ratios obtained with the new DNA chip were validated against those obtained with quantitative real-time PCR assays. This novel microarray technology would be a powerful tool for monitoring gene expression profiles, especially for clinical diagnosis.     

A survey of the cellular responses in Pseudomonas putida KT2440 growing in sterilized soil by microarray analysis

Genome-wide scanning of gene expression by microarray techniques was successfully performed on RNA extracted from sterilized soil inoculated with Pseudomonas putida KT2440/pSL1, which contains a chloroaromatic degrading plasmid, in the presence or absence of 3-chlorobenzoic acid (3CB). The genes showing significant changes in their expression in both the triplicate-microarray analysis using amplified RNA and the single-microarray analysis using unamplified RNA were investigated. Pathway analysis revealed that the benzoate degradation pathway underwent the most significant changes following treatment with 3CB. Analysis based on categorization of differentially expressed genes against 3CB revealed new findings about the cellular responses of the bacteria to 3CB. Genes specifically involved in the transport of 3CB were upregulated, including a K(+)/H(+) antiporter complex, a universal stress protein, two cytochrome P450 proteins and an efflux transporter. The downregulated expression of several genes involved in carbon metabolism and the genes belonging to a prophage in the presence of 3CB was observed. This study demonstrated the applicability of the method of soil RNA extraction for microarray analysis of gene expression in bacteria growing in sterilized soil.     

Identification of Chromosomal Errors in Human Preimplantation Embryos with Oligonucleotide DNA Microarray

A previous study comparing the performance of different platforms for DNA microarray found that the oligonucleotide (oligo) microarray platform containing 385K isothermal probes had the best performance when evaluating dosage sensitivity, precision, specificity, sensitivity and copy number variations border definition. Although oligo microarray platform has been used in some research fields and clinics, it has not been used for aneuploidy screening in human embryos. The present study was designed to use this new microarray platform for preimplantation genetic screening in the human. A total of 383 blastocysts from 72 infertility patients with either advanced maternal age or with previous miscarriage were analyzed after biopsy and microarray. Euploid blastocysts were transferred to patients and clinical pregnancy and implantation rates were measured. Chromosomes in some aneuploid blastocysts were further analyzed by fluorescence in-situ hybridization (FISH) to evaluate accuracy of the results. We found that most (58.1%) of the blastocysts had chromosomal abnormalities that included single or multiple gains and/or losses of chromosome(s), partial chromosome deletions and/or duplications in both euploid and aneuploid embryos. Transfer of normal euploid blastocysts in 34 cycles resulted in 58.8% clinical pregnancy and 54.4% implantation rates. Examination of abnormal blastocysts by FISH showed that all embryos had matching results comparing microarray and FISH analysis. The present study indicates that oligo microarray conducted with a higher resolution and a greater number of probes is able to detect not only aneuploidy, but also minor chromosomal abnormalities, such as partial chromosome deletion and/or duplication in human embryos. Preimplantation genetic screening of the aneuploidy by DNA microarray is an advanced technology used to select embryos for transfer and improved embryo implantation can be obtained after transfer of the screened normal embryos.