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Sample records for automated rna extraction

  1. Automated RNA Extraction and Purification for Multiplexed Pathogen Detection

    SciTech Connect

    Bruzek, Amy K.; Bruckner-Lea, Cindy J.

    2005-01-01

    Pathogen detection has become an extremely important part of our nation?s defense in this post 9/11 world where the threat of bioterrorist attacks are a grim reality. When a biological attack takes place, response time is critical. The faster the biothreat is assessed, the faster countermeasures can be put in place to protect the health of the general public. Today some of the most widely used methods for detecting pathogens are either time consuming or not reliable [1]. Therefore, a method that can detect multiple pathogens that is inherently reliable, rapid, automated and field portable is needed. To that end, we are developing automated fluidics systems for the recovery, cleanup, and direct labeling of community RNA from suspect environmental samples. The advantage of using RNA for detection is that there are multiple copies of mRNA in a cell, whereas there are normally only one or two copies of DNA [2]. Because there are multiple copies of mRNA in a cell for highly expressed genes, no amplification of the genetic material may be necessary, and thus rapid and direct detection of only a few cells may be possible [3]. This report outlines the development of both manual and automated methods for the extraction and purification of mRNA. The methods were evaluated using cell lysates from Escherichia coli 25922 (nonpathogenic), Salmonella typhimurium (pathogenic), and Shigella spp (pathogenic). Automated RNA purification was achieved using a custom sequential injection fluidics system consisting of a syringe pump, a multi-port valve and a magnetic capture cell. mRNA was captured using silica coated superparamagnetic beads that were trapped in the tubing by a rare earth magnet. RNA was detected by gel electrophoresis and/or by hybridization of the RNA to microarrays. The versatility of the fluidics systems and the ability to automate these systems allows for quick and easy processing of samples and eliminates the need for an experienced operator.

  2. Automated motif extraction and classification in RNA tertiary structures

    PubMed Central

    Djelloul, Mahassine; Denise, Alain

    2008-01-01

    We used a novel graph-based approach to extract RNA tertiary motifs. We cataloged them all and clustered them using an innovative graph similarity measure. We applied our method to three widely studied structures: Haloarcula marismortui 50S (H.m 50S), Escherichia coli 50S (E. coli 50S), and Thermus thermophilus 16S (T.th 16S) RNAs. We identified 10 known motifs without any prior knowledge of their shapes or positions. We additionally identified four putative new motifs. PMID:18957493

  3. Automated serial extraction of DNA and RNA from biobanked tissue specimens.

    PubMed

    Mathot, Lucy; Wallin, Monica; Sjöblom, Tobias

    2013-08-19

    With increasing biobanking of biological samples, methods for large scale extraction of nucleic acids are in demand. The lack of such techniques designed for extraction from tissues results in a bottleneck in downstream genetic analyses, particularly in the field of cancer research. We have developed an automated procedure for tissue homogenization and extraction of DNA and RNA into separate fractions from the same frozen tissue specimen. A purpose developed magnetic bead based technology to serially extract both DNA and RNA from tissues was automated on a Tecan Freedom Evo robotic workstation. 864 fresh-frozen human normal and tumor tissue samples from breast and colon were serially extracted in batches of 96 samples. Yields and quality of DNA and RNA were determined. The DNA was evaluated in several downstream analyses, and the stability of RNA was determined after 9 months of storage. The extracted DNA performed consistently well in processes including PCR-based STR analysis, HaloPlex selection and deep sequencing on an Illumina platform, and gene copy number analysis using microarrays. The RNA has performed well in RT-PCR analyses and maintains integrity upon storage. The technology described here enables the processing of many tissue samples simultaneously with a high quality product and a time and cost reduction for the user. This reduces the sample preparation bottleneck in cancer research. The open automation format also enables integration with upstream and downstream devices for automated sample quantitation or storage.

  4. Automated serial extraction of DNA and RNA from biobanked tissue specimens

    PubMed Central

    2013-01-01

    Background With increasing biobanking of biological samples, methods for large scale extraction of nucleic acids are in demand. The lack of such techniques designed for extraction from tissues results in a bottleneck in downstream genetic analyses, particularly in the field of cancer research. We have developed an automated procedure for tissue homogenization and extraction of DNA and RNA into separate fractions from the same frozen tissue specimen. A purpose developed magnetic bead based technology to serially extract both DNA and RNA from tissues was automated on a Tecan Freedom Evo robotic workstation. Results 864 fresh-frozen human normal and tumor tissue samples from breast and colon were serially extracted in batches of 96 samples. Yields and quality of DNA and RNA were determined. The DNA was evaluated in several downstream analyses, and the stability of RNA was determined after 9 months of storage. The extracted DNA performed consistently well in processes including PCR-based STR analysis, HaloPlex selection and deep sequencing on an Illumina platform, and gene copy number analysis using microarrays. The RNA has performed well in RT-PCR analyses and maintains integrity upon storage. Conclusions The technology described here enables the processing of many tissue samples simultaneously with a high quality product and a time and cost reduction for the user. This reduces the sample preparation bottleneck in cancer research. The open automation format also enables integration with upstream and downstream devices for automated sample quantitation or storage. PMID:23957867

  5. Automated microfluidic DNA/RNA extraction with both disposable and reusable components

    NASA Astrophysics Data System (ADS)

    Kim, Jungkyu; Johnson, Michael; Hill, Parker; Sonkul, Rahul S.; Kim, Jongwon; Gale, Bruce K.

    2012-01-01

    An automated microfluidic nucleic extraction system was fabricated with a multilayer polydimethylsiloxane (PDMS) structure that consists of sample wells, microvalves, a micropump and a disposable microfluidic silica cartridge. Both the microvalves and micropump structures were fabricated in a single layer and are operated pneumatically using a 100 µm PDMS membrane. To fabricate the disposable microfluidic silica cartridge, two-cavity structures were made in a PDMS replica to fit the stacked silica membranes. A handheld controller for the microvalves and pumps was developed to enable system automation. With purified ribonucleic acid (RNA), whole blood and E. coli samples, the automated microfluidic nucleic acid extraction system was validated with a guanidine-based solid phase extraction procedure. An extraction efficiency of ~90% for deoxyribonucleic acid (DNA) and ~54% for RNA was obtained in 12 min from whole blood and E. coli samples, respectively. In addition, the same quantity and quality of extracted DNA was confirmed by polymerase chain reaction (PCR) amplification. The PCR also presented the appropriate amplification and melting profiles. Automated, programmable fluid control and physical separation of the reusable components and the disposable components significantly decrease the assay time and manufacturing cost and increase the flexibility and compatibility of the system with downstream components.

  6. Automated Device for Asynchronous Extraction of RNA, DNA, or Protein Biomarkers from Surrogate Patient Samples.

    PubMed

    Bitting, Anna L; Bordelon, Hali; Baglia, Mark L; Davis, Keersten M; Creecy, Amy E; Short, Philip A; Albert, Laura E; Karhade, Aditya V; Wright, David W; Haselton, Frederick R; Adams, Nicholas M

    2016-12-01

    Many biomarker-based diagnostic methods are inhibited by nontarget molecules in patient samples, necessitating biomarker extraction before detection. We have developed a simple device that purifies RNA, DNA, or protein biomarkers from complex biological samples without robotics or fluid pumping. The device design is based on functionalized magnetic beads, which capture biomarkers and remove background biomolecules by magnetically transferring the beads through processing solutions arrayed within small-diameter tubing. The process was automated by wrapping the tubing around a disc-like cassette and rotating it past a magnet using a programmable motor. This device recovered biomarkers at ~80% of the operator-dependent extraction method published previously. The device was validated by extracting biomarkers from a panel of surrogate patient samples containing clinically relevant concentrations of (1) influenza A RNA in nasal swabs, (2) Escherichia coli DNA in urine, (3) Mycobacterium tuberculosis DNA in sputum, and (4) Plasmodium falciparum protein and DNA in blood. The device successfully extracted each biomarker type from samples representing low levels of clinically relevant infectivity (i.e., 7.3 copies/µL of influenza A RNA, 405 copies/µL of E. coli DNA, 0.22 copies/µL of TB DNA, 167 copies/µL of malaria parasite DNA, and 2.7 pM of malaria parasite protein). © 2015 Society for Laboratory Automation and Screening.

  7. Automation of DNA and miRNA co-extraction for miRNA-based identification of human body fluids and tissues.

    PubMed

    Kulstein, Galina; Marienfeld, Ralf; Miltner, Erich; Wiegand, Peter

    2016-10-01

    In the last years, microRNA (miRNA) analysis came into focus in the field of forensic genetics. Yet, no standardized and recommendable protocols for co-isolation of miRNA and DNA from forensic relevant samples have been developed so far. Hence, this study evaluated the performance of an automated Maxwell® 16 System-based strategy (Promega) for co-extraction of DNA and miRNA from forensically relevant (blood and saliva) samples compared to (semi-)manual extraction methods. Three procedures were compared on the basis of recovered quantity of DNA and miRNA (as determined by real-time PCR and Bioanalyzer), miRNA profiling (shown by Cq values and extraction efficiency), STR profiles, duration, contamination risk and handling. All in all, the results highlight that the automated co-extraction procedure yielded the highest miRNA and DNA amounts from saliva and blood samples compared to both (semi-)manual protocols. Also, for aged and genuine samples of forensically relevant traces the miRNA and DNA yields were sufficient for subsequent downstream analysis. Furthermore, the strategy allows miRNA extraction only in cases where it is relevant to obtain additional information about the sample type. Besides, this system enables flexible sample throughput and labor-saving sample processing with reduced risk of cross-contamination.

  8. Comparative evaluation of commercially available manual and automated nucleic acid extraction methods for rotavirus RNA detection in stools.

    PubMed

    Esona, Mathew D; McDonald, Sharla; Kamili, Shifaq; Kerin, Tara; Gautam, Rashi; Bowen, Michael D

    2013-12-01

    Rotaviruses are a major cause of viral gastroenteritis in children. For accurate and sensitive detection of rotavirus RNA from stool samples by reverse transcription-polymerase chain reaction (RT-PCR), the extraction process must be robust. However, some extraction methods may not remove the strong RT-PCR inhibitors known to be present in stool samples. The objective of this study was to evaluate and compare the performance of six extraction methods used commonly for extraction of rotavirus RNA from stool, which have never been formally evaluated: the MagNA Pure Compact, KingFisher Flex and NucliSENS easyMAG instruments, the NucliSENS miniMAG semi-automated system, and two manual purification kits, the QIAamp Viral RNA kit and a modified RNaid kit. Using each method, total nucleic acid or RNA was extracted from eight rotavirus-positive stool samples with enzyme immunoassay optical density (EIA OD) values ranging from 0.176 to 3.098. Extracts prepared using the MagNA Pure Compact instrument yielded the most consistent results by qRT-PCR and conventional RT-PCR. When extracts prepared from a dilution series were extracted by the 6 methods and tested, rotavirus RNA was detected in all samples by qRT-PCR but by conventional RT-PCR testing, only the MagNA Pure Compact and KingFisher Flex extracts were positive in all cases. RT-PCR inhibitors were detected in extracts produced with the QIAamp Viral RNA Mini kit. The findings of this study should prove useful for selection of extraction methods to be incorporated into future rotavirus detection and genotyping protocols.

  9. Automated Purification and Suspension Array Detection of 16S rRNA from Soil and Sediment Extracts Using Tunable Surface Microparticles

    SciTech Connect

    Chandler, Darrell P.; Jarrell, Ann E.

    2004-05-01

    Autonomous, field-deployable molecular detection systems require seamless integration of complex biochemical solutions and physical or mechanical processing steps. In an attempt to simplify the fluidic requirements for integrated biodetection systems, we used tunable surface microparticles both as an rRNA affinity purification resin in a renewable microcolumn sample preparation system and as the sensor surface in a flow cytometer detector. The tunable surface detection limits in both low- and high-salt buffers were 1 ng of total RNA (~104 cell equivalents) in 15-min test tube hybridizations and 10 ng of total RNA (~105 cell equivalents) in hybridizations with the automated system (30-s contact time). RNA fragmentation was essential for achieving tunable surface suspension array specificity. Chaperone probes reduced but did not completely eliminate cross-hybridization, even with probes sharing <50% identity to target sequences. Nonpurified environmental extracts did not irreparably affect our ability to classify color-coded microparticles, but residual environmental constituents significantly quenched the Alexa-532 reporter fluor. Modulating surface charge did not influence the interaction of soluble environmental contaminants with conjugated beads. The automated system greatly reduced the effects of fluorescence quenching, especially in the soil background. The automated system was as efficacious as manual methods for simultaneous sample purification, hybridization, and washing prior to flow cytometry detection. The implications of unexpected target cross-hybridization and fluorescence quenching are discussed relative to the design and implementation of an integrated microbial monitoring system.

  10. Automated extraction of DNA and RNA from a single formalin-fixed paraffin-embedded tissue section for analysis of both single-nucleotide polymorphisms and mRNA expression.

    PubMed

    Hennig, Guido; Gehrmann, Mathias; Stropp, Udo; Brauch, Hiltrud; Fritz, Peter; Eichelbaum, Michel; Schwab, Matthias; Schroth, Werner

    2010-12-01

    There is an increasing need for the identification of both DNA and RNA biomarkers from pathodiagnostic formalin-fixed paraffin-embedded (FFPE) tissue samples for the exploration of individualized therapy strategies in cancer. We investigated a fully automated, xylene-free nucleic acid extraction method for the simultaneous analysis of RNA and DNA biomarkers related to breast cancer. We copurified both RNA and DNA from a single 10-μm section of 210 paired samples of FFPE tumor and adjacent normal tissues (1-25 years of archival time) using a fully automated extraction method. Half of the eluate was DNase I digested for mRNA expression analysis performed by using reverse-transcription quantitative PCR for the genes estrogen receptor 1 (ESR1), progesterone receptor (PGR), v-erb-b2 erythroblastic leukemia viral oncogene homolog 2, neuro/glioblastoma derived oncogene homolog (avian) (ERBB2), epoxide hydrolase 1 (EPHX1), baculoviral IAP repeat-containing 5 (BIRC5), matrix metallopeptidase 7 (MMP7), vascular endothelial growth factor A (VEGFA), and topoisomerase (DNA) II alpha 170kDa (TOP2A). The remaining undigested aliquot was used for the analysis of 7 single-nucleotide polymorphisms (SNPs) by MALDI-TOF mass spectrometry. In 208 of 210 samples (99.0%) the protocol yielded robust quantification-cycle values for both RNA and DNA normalization. Expression of the 8 breast cancer genes was detected in 81%-100% of tumor tissues and 21%-100% of normal tissues. The 7 SNPs were successfully genotyped in 91%-97% of tumor and 94%-97% of normal tissues. Allele concordance between tumor and normal tissue was 98.9%-99.5%. This fully automated process allowed an efficient simultaneous extraction of both RNA and DNA from a single FFPE section and subsequent dual analysis of selected genes. High gene expression and genotyping detection rates demonstrate the feasibility of molecular profiling from limited archival patient samples.

  11. A comparison of commercially-available automated and manual extraction kits for the isolation of total RNA from small tissue samples.

    PubMed

    Sellin Jeffries, Marlo K; Kiss, Andor J; Smith, Austin W; Oris, James T

    2014-11-14

    This study compared the performance of five commercially available kits in extracting total RNA from small eukaryotic tissue samples (<15 mg). Total RNA was isolated from fathead minnow (Pimephales promelas) tissues (spleen, blood, kidney, embryo, and larvae) using the Qiagen RNeasy® Plus Mini, Qiagen RNeasy® Plus Universal, Promega Maxwell® 16 LEV simplyRNA, Ambion MagMAX™-96 and Promega SimplyRNA HT kits. Kit performance was evaluated via measures of RNA quantity (e.g., total RNA amount) and quality (e.g., ratio of absorbance at 260 and 280 nm, RNA integrity number (RIN), presence of gDNA). With the exception of embryos, each kit generally extracted ≥5 μg of total RNA from each sample. With regard to RNA quality, the RINs of RNA samples isolated via the Plus Mini and Maxwell® 16 kits were consistently higher than those of samples extracted via the remaining three kits and for all tissues, these kits produced intact RNA with average RIN values ≥7. The Plus Universal and SimplyRNA HT kits produced moderately degraded (RIN values <7, but ≥5), while the RNA recovered via the MagMAX™ kit tended to exhibit a high degree of degradation (RIN values <5). Each kit was generally capable of extracting the amount of RNA required for most downstream gene expression applications suggesting that RNA yield is unlikely to be a limiting factor for any of the kits evaluated. However, differences in the quality of RNA extracted via each of the kits indicate that these kits may differ in their ability to yield RNA acceptable for some applications. Overall, the findings of this study demonstrate that there are practical differences between commercially available RNA extraction kits that should be taken into account when selecting extraction methods to be used for isolating RNA designated for gene expression analysis.

  12. Comparison of the MagNA pure LC automated system and the RiboPure-Blood RNA manual method for RNA extraction from multiple myeloma bone marrow samples conserved in an RNA stabilizer.

    PubMed

    Garcia-Effron, G; Gamarra, S; Crooke, A; Martínez-Sánchez, P; Lahuerta, J; Martínez-López, J

    2007-04-01

    A total of 62 frozen bone marrow specimens conserved in RNA later (Ambion) were processed using two different extraction methods, the MagNA Pure LC system (MAG; Roche) and the manual RiboPure-Blood RNA method (RIBO; Ambion); Beta glucoronidase RNA (GUS) was amplified by LightCycler PCR to evaluate the quality of both extraction procedures. Less than 1000 GUS copies/ml was detected in 26 of 62 specimens (41.94%) processed by MAG and in five of 62 specimens (8.06%) processed by RIBO. Moreover, RNA recovery from the 62 specimens by MAG is, on average, 2.91 cycle threshold-fold higher than RIBO (P = 0.0008). Furthermore, we compared the extraction times and reagent costs of both methods. In conclusion, RNA extraction using MAG is faster to process 32 samples and less expensive than RIBO but it is not sensitive enough to be employed for research purpose in our laboratory.

  13. High-throughput, automated extraction of DNA and RNA from clinical samples using TruTip technology on common liquid handling robots.

    PubMed

    Holmberg, Rebecca C; Gindlesperger, Alissa; Stokes, Tinsley; Brady, Dane; Thakore, Nitu; Belgrader, Philip; Cooney, Christopher G; Chandler, Darrell P

    2013-06-11

    TruTip is a simple nucleic acid extraction technology whereby a porous, monolithic binding matrix is inserted into a pipette tip. The geometry of the monolith can be adapted for specific pipette tips ranging in volume from 1.0 to 5.0 ml. The large porosity of the monolith enables viscous or complex samples to readily pass through it with minimal fluidic backpressure. Bi-directional flow maximizes residence time between the monolith and sample, and enables large sample volumes to be processed within a single TruTip. The fundamental steps, irrespective of sample volume or TruTip geometry, include cell lysis, nucleic acid binding to the inner pores of the TruTip monolith, washing away unbound sample components and lysis buffers, and eluting purified and concentrated nucleic acids into an appropriate buffer. The attributes and adaptability of TruTip are demonstrated in three automated clinical sample processing protocols using an Eppendorf epMotion 5070, Hamilton STAR and STARplus liquid handling robots, including RNA isolation from nasopharyngeal aspirate, genomic DNA isolation from whole blood, and fetal DNA extraction and enrichment from large volumes of maternal plasma (respectively).

  14. High-throughput, Automated Extraction of DNA and RNA from Clinical Samples using TruTip Technology on Common Liquid Handling Robots

    PubMed Central

    Holmberg, Rebecca C.; Gindlesperger, Alissa; Stokes, Tinsley; Brady, Dane; Thakore, Nitu; Belgrader, Philip; Cooney, Christopher G.; Chandler, Darrell P.

    2013-01-01

    TruTip is a simple nucleic acid extraction technology whereby a porous, monolithic binding matrix is inserted into a pipette tip. The geometry of the monolith can be adapted for specific pipette tips ranging in volume from 1.0 to 5.0 ml. The large porosity of the monolith enables viscous or complex samples to readily pass through it with minimal fluidic backpressure. Bi-directional flow maximizes residence time between the monolith and sample, and enables large sample volumes to be processed within a single TruTip. The fundamental steps, irrespective of sample volume or TruTip geometry, include cell lysis, nucleic acid binding to the inner pores of the TruTip monolith, washing away unbound sample components and lysis buffers, and eluting purified and concentrated nucleic acids into an appropriate buffer. The attributes and adaptability of TruTip are demonstrated in three automated clinical sample processing protocols using an Eppendorf epMotion 5070, Hamilton STAR and STARplus liquid handling robots, including RNA isolation from nasopharyngeal aspirate, genomic DNA isolation from whole blood, and fetal DNA extraction and enrichment from large volumes of maternal plasma (respectively). PMID:23793016

  15. Integrated DNA and RNA extraction and purification on an automated microfluidic cassette from bacterial and viral pathogens causing community-acquired lower respiratory tract infections.

    PubMed

    Van Heirstraeten, Liesbet; Spang, Peter; Schwind, Carmen; Drese, Klaus S; Ritzi-Lehnert, Marion; Nieto, Benjamin; Camps, Marta; Landgraf, Bryan; Guasch, Francesc; Corbera, Antoni Homs; Samitier, Josep; Goossens, Herman; Malhotra-Kumar, Surbhi; Roeser, Tina

    2014-05-07

    In this paper, we describe the development of an automated sample preparation procedure for etiological agents of community-acquired lower respiratory tract infections (CA-LRTI). The consecutive assay steps, including sample re-suspension, pre-treatment, lysis, nucleic acid purification, and concentration, were integrated into a microfluidic lab-on-a-chip (LOC) cassette that is operated hands-free by a demonstrator setup, providing fluidic and valve actuation. The performance of the assay was evaluated on viral and Gram-positive and Gram-negative bacterial broth cultures previously sampled using a nasopharyngeal swab. Sample preparation on the microfluidic cassette resulted in higher or similar concentrations of pure bacterial DNA or viral RNA compared to manual benchtop experiments. The miniaturization and integration of the complete sample preparation procedure, to extract purified nucleic acids from real samples of CA-LRTI pathogens to, and above, lab quality and efficiency, represent important steps towards its application in a point-of-care test (POCT) for rapid diagnosis of CA-LRTI.

  16. Automated Extraction of Flow Features

    NASA Technical Reports Server (NTRS)

    Dorney, Suzanne (Technical Monitor); Haimes, Robert

    2004-01-01

    Computational Fluid Dynamics (CFD) simulations are routinely performed as part of the design process of most fluid handling devices. In order to efficiently and effectively use the results of a CFD simulation, visualization tools are often used. These tools are used in all stages of the CFD simulation including pre-processing, interim-processing, and post-processing, to interpret the results. Each of these stages requires visualization tools that allow one to examine the geometry of the device, as well as the partial or final results of the simulation. An engineer will typically generate a series of contour and vector plots to better understand the physics of how the fluid is interacting with the physical device. Of particular interest are detecting features such as shocks, recirculation zones, and vortices (which will highlight areas of stress and loss). As the demand for CFD analyses continues to increase the need for automated feature extraction capabilities has become vital. In the past, feature extraction and identification were interesting concepts, but not required in understanding the physics of a steady flow field. This is because the results of the more traditional tools like; iso-surface, cuts and streamlines, were more interactive and easily abstracted so they could be represented to the investigator. These tools worked and properly conveyed the collected information at the expense of a great deal of interaction. For unsteady flow-fields, the investigator does not have the luxury of spending time scanning only one "snapshot" of the simulation. Automated assistance is required in pointing out areas of potential interest contained within the flow. This must not require a heavy compute burden (the visualization should not significantly slow down the solution procedure for (co-processing environments). Methods must be developed to abstract the feature of interest and display it in a manner that physically makes sense.

  17. Automated Extraction of Flow Features

    NASA Technical Reports Server (NTRS)

    Dorney, Suzanne (Technical Monitor); Haimes, Robert

    2005-01-01

    Computational Fluid Dynamics (CFD) simulations are routinely performed as part of the design process of most fluid handling devices. In order to efficiently and effectively use the results of a CFD simulation, visualization tools are often used. These tools are used in all stages of the CFD simulation including pre-processing, interim-processing, and post-processing, to interpret the results. Each of these stages requires visualization tools that allow one to examine the geometry of the device, as well as the partial or final results of the simulation. An engineer will typically generate a series of contour and vector plots to better understand the physics of how the fluid is interacting with the physical device. Of particular interest are detecting features such as shocks, re-circulation zones, and vortices (which will highlight areas of stress and loss). As the demand for CFD analyses continues to increase the need for automated feature extraction capabilities has become vital. In the past, feature extraction and identification were interesting concepts, but not required in understanding the physics of a steady flow field. This is because the results of the more traditional tools like; isc-surface, cuts and streamlines, were more interactive and easily abstracted so they could be represented to the investigator. These tools worked and properly conveyed the collected information at the expense of a great deal of interaction. For unsteady flow-fields, the investigator does not have the luxury of spending time scanning only one "snapshot" of the simulation. Automated assistance is required in pointing out areas of potential interest contained within the flow. This must not require a heavy compute burden (the visualization should not significantly slow down the solution procedure for co-processing environments). Methods must be developed to abstract the feature of interest and display it in a manner that physically makes sense.

  18. Automated modeling of RNA 3D structure.

    PubMed

    Rother, Kristian; Rother, Magdalena; Skiba, Pawel; Bujnicki, Janusz M

    2014-01-01

    This chapter gives an overview over the current methods for automated modeling of RNA structures, with emphasis on template-based methods. The currently used approaches to RNA modeling are presented with a side view on the protein world, where many similar ideas have been used. Two main programs for automated template-based modeling are presented: ModeRNA assembling structures from fragments and MacroMoleculeBuilder performing a simulation to satisfy spatial restraints. Both approaches have in common that they require an alignment of the target sequence to a known RNA structure that is used as a modeling template. As a way to find promising template structures and to align the target and template sequences, we propose a pipeline combining the ParAlign and Infernal programs on RNA family data from Rfam. We also briefly summarize template-free methods for RNA 3D structure prediction. Typically, RNA structures generated by automated modeling methods require local or global optimization. Thus, we also discuss methods that can be used for local or global refinement of RNA structures.

  19. Automated DNA extraction from pollen in honey.

    PubMed

    Guertler, Patrick; Eicheldinger, Adelina; Muschler, Paul; Goerlich, Ottmar; Busch, Ulrich

    2014-04-15

    In recent years, honey has become subject of DNA analysis due to potential risks evoked by microorganisms, allergens or genetically modified organisms. However, so far, only a few DNA extraction procedures are available, mostly time-consuming and laborious. Therefore, we developed an automated DNA extraction method from pollen in honey based on a CTAB buffer-based DNA extraction using the Maxwell 16 instrument and the Maxwell 16 FFS Nucleic Acid Extraction System, Custom-Kit. We altered several components and extraction parameters and compared the optimised method with a manual CTAB buffer-based DNA isolation method. The automated DNA extraction was faster and resulted in higher DNA yield and sufficient DNA purity. Real-time PCR results obtained after automated DNA extraction are comparable to results after manual DNA extraction. No PCR inhibition was observed. The applicability of this method was further successfully confirmed by analysis of different routine honey samples. Copyright © 2013 Elsevier Ltd. All rights reserved.

  20. RCrane: semi-automated RNA model building.

    PubMed

    Keating, Kevin S; Pyle, Anna Marie

    2012-08-01

    RNA crystals typically diffract to much lower resolutions than protein crystals. This low-resolution diffraction results in unclear density maps, which cause considerable difficulties during the model-building process. These difficulties are exacerbated by the lack of computational tools for RNA modeling. Here, RCrane, a tool for the partially automated building of RNA into electron-density maps of low or intermediate resolution, is presented. This tool works within Coot, a common program for macromolecular model building. RCrane helps crystallographers to place phosphates and bases into electron density and then automatically predicts and builds the detailed all-atom structure of the traced nucleotides. RCrane then allows the crystallographer to review the newly built structure and select alternative backbone conformations where desired. This tool can also be used to automatically correct the backbone structure of previously built nucleotides. These automated corrections can fix incorrect sugar puckers, steric clashes and other structural problems.

  1. RCrane: semi-automated RNA model building

    SciTech Connect

    Keating, Kevin S.; Pyle, Anna Marie

    2012-08-01

    RCrane is a new tool for the partially automated building of RNA crystallographic models into electron-density maps of low or intermediate resolution. This tool helps crystallographers to place phosphates and bases into electron density and then automatically predicts and builds the detailed all-atom structure of the traced nucleotides. RNA crystals typically diffract to much lower resolutions than protein crystals. This low-resolution diffraction results in unclear density maps, which cause considerable difficulties during the model-building process. These difficulties are exacerbated by the lack of computational tools for RNA modeling. Here, RCrane, a tool for the partially automated building of RNA into electron-density maps of low or intermediate resolution, is presented. This tool works within Coot, a common program for macromolecular model building. RCrane helps crystallographers to place phosphates and bases into electron density and then automatically predicts and builds the detailed all-atom structure of the traced nucleotides. RCrane then allows the crystallographer to review the newly built structure and select alternative backbone conformations where desired. This tool can also be used to automatically correct the backbone structure of previously built nucleotides. These automated corrections can fix incorrect sugar puckers, steric clashes and other structural problems.

  2. Evaluation of DNA and RNA extraction methods.

    PubMed

    Edwin Shiaw, C S; Shiran, M S; Cheah, Y K; Tan, G C; Sabariah, A R

    2010-06-01

    This study was done to evaluate various DNA and RNA extractions from archival FFPE tissues. A total of 30 FFPE blocks from the years of 2004 to 2006 were assessed with each modified and adapted method. Extraction protocols evaluated include the modified enzymatic extraction method (Method A), Chelex-100 extraction method (Method B), heat-induced retrieval in alkaline solution extraction method (Methods C and D) and one commercial FFPE DNA Extraction kit (Qiagen, Crawley, UK). For RNA extraction, 2 extraction protocols were evaluated including the enzymatic extraction method (Method 1), and Chelex-100 RNA extraction method (Method 2). Results show that the modified enzymatic extraction method (Method A) is an efficient DNA extraction protocol, while for RNA extraction, the enzymatic method (Method 1) and the Chelex-100 RNA extraction method (Method 2) are equally efficient RNA extraction protocols.

  3. Avian influenza virus RNA extraction

    USDA-ARS?s Scientific Manuscript database

    The efficient extraction and purification of viral RNA is critical for down-stream molecular applications whether it is the sensitive and specific detection of virus in clinical samples, virus gene cloning and expression, or quantification of avian influenza (AI) virus by molecular methods from expe...

  4. Automated classification of RNA 3D motifs and the RNA 3D Motif Atlas

    PubMed Central

    Petrov, Anton I.; Zirbel, Craig L.; Leontis, Neocles B.

    2013-01-01

    The analysis of atomic-resolution RNA three-dimensional (3D) structures reveals that many internal and hairpin loops are modular, recurrent, and structured by conserved non-Watson–Crick base pairs. Structurally similar loops define RNA 3D motifs that are conserved in homologous RNA molecules, but can also occur at nonhomologous sites in diverse RNAs, and which often vary in sequence. To further our understanding of RNA motif structure and sequence variability and to provide a useful resource for structure modeling and prediction, we present a new method for automated classification of internal and hairpin loop RNA 3D motifs and a new online database called the RNA 3D Motif Atlas. To classify the motif instances, a representative set of internal and hairpin loops is automatically extracted from a nonredundant list of RNA-containing PDB files. Their structures are compared geometrically, all-against-all, using the FR3D program suite. The loops are clustered into motif groups, taking into account geometric similarity and structural annotations and making allowance for a variable number of bulged bases. The automated procedure that we have implemented identifies all hairpin and internal loop motifs previously described in the literature. All motif instances and motif groups are assigned unique and stable identifiers and are made available in the RNA 3D Motif Atlas (http://rna.bgsu.edu/motifs), which is automatically updated every four weeks. The RNA 3D Motif Atlas provides an interactive user interface for exploring motif diversity and tools for programmatic data access. PMID:23970545

  5. Automated classification of RNA 3D motifs and the RNA 3D Motif Atlas.

    PubMed

    Petrov, Anton I; Zirbel, Craig L; Leontis, Neocles B

    2013-10-01

    The analysis of atomic-resolution RNA three-dimensional (3D) structures reveals that many internal and hairpin loops are modular, recurrent, and structured by conserved non-Watson-Crick base pairs. Structurally similar loops define RNA 3D motifs that are conserved in homologous RNA molecules, but can also occur at nonhomologous sites in diverse RNAs, and which often vary in sequence. To further our understanding of RNA motif structure and sequence variability and to provide a useful resource for structure modeling and prediction, we present a new method for automated classification of internal and hairpin loop RNA 3D motifs and a new online database called the RNA 3D Motif Atlas. To classify the motif instances, a representative set of internal and hairpin loops is automatically extracted from a nonredundant list of RNA-containing PDB files. Their structures are compared geometrically, all-against-all, using the FR3D program suite. The loops are clustered into motif groups, taking into account geometric similarity and structural annotations and making allowance for a variable number of bulged bases. The automated procedure that we have implemented identifies all hairpin and internal loop motifs previously described in the literature. All motif instances and motif groups are assigned unique and stable identifiers and are made available in the RNA 3D Motif Atlas (http://rna.bgsu.edu/motifs), which is automatically updated every four weeks. The RNA 3D Motif Atlas provides an interactive user interface for exploring motif diversity and tools for programmatic data access.

  6. Acceleration of Automated HI Source Extraction

    NASA Astrophysics Data System (ADS)

    Badenhorst, S. J.; Blyth, S.; Kuttel, M. M.

    2013-10-01

    We aim to enable fast automated extraction of neutral hydrogen (HI) sources from large survey data sets. This requires both handling the large files (>5 TB) to be produced by next-generation interferometers and acceleration of the source extraction algorithm. We develop an efficient multithreaded implementation of the A'Trous wavelet reconstruction algorithm, which we evaluate against the serial implementation in the DUCHAMP package. We also evaluate three memory management libraries (Mmap, Boost and Stxxl) that enable processing of data files too large to fit into main memory, to establish which provides the best performance.

  7. Avian influenza virus RNA extraction.

    PubMed

    Spackman, Erica; Lee, Scott A

    2014-01-01

    The efficient extraction and purification of viral RNA is critical for down-stream molecular applications whether it is the sensitive and specific detection of virus in clinical samples, virus gene cloning and expression, or quantification of avian influenza (AI) virus by molecular methods from experimentally infected birds. Samples can generally be divided into two types; enriched (e.g. virus stocks) and clinical. Clinical type samples, which may be tissues or swab material, are the most difficult to process due to the complex sample composition and possibly low virus titers. In this chapter two well established procedures for the isolation of AI virus RNA from common clinical specimen types and enriched virus stocks for further molecular applications will be presented.

  8. Comparison of automated and manual purification of total RNA for mRNA-based identification of body fluids.

    PubMed

    Akutsu, Tomoko; Kitayama, Tetsushi; Watanabe, Ken; Sakurada, Koichi

    2015-01-01

    Silica column-based RNA purification procedures have widespread use in mRNA profiling for body fluid identification in forensic samples. Also, automated RNA purification systems employing magnetic bead technology have recently become available. In this preliminary study, to ascertain which RNA purification technology is more suitable for the identification of body fluids by real-time reverse transcription polymerase chain reaction (RT-PCR), comparative analyses of the yield and quality of total RNA were performed between automated purification using an EZ1 Advanced Instrument and manual purification using an RNeasy Mini Kit. The yield and size distribution of total RNA were compared by gene expression analysis of two different sized fragments of the β-actin gene. In addition, the relative amounts of several target genes were compared between the purification methods, and the integrity of total RNA was determined by chip-based electrophoresis. The results of this study suggest that RNeasy can purify higher-quality RNA as compared with automated purification using EZ1. The sensitivity of the RT-PCR analysis, however, was higher in the EZ1-purified samples, likely due to the relative efficiency of EZ1 in extracting short-length RNA from degraded samples. We also show that the quantification of relative levels of body fluid-specific genes could be influenced by the purification procedure. Our results indicate that although use of high-quality RNA is generally required for reproducible results in gene expression analysis, the forensic relevance of short RNA fragments in highly degraded samples cannot be ruled out. Furthermore, our results suggest that automated purification procedures as well as silica column-based manual purification procedures can be used for mRNA-based body fluid identification in forensic samples.

  9. Rapid, simple influenza RNA extraction from nasopharyngeal samples

    PubMed Central

    Chandler, Darrell P.; Griesemer, Sara B.; Cooney, Christopher G.; Holmberg, Rebecca; Thakore, Nitu; Mokhiber, Becca; Belgrader, Phillip; Knickerbocker, Christopher; Schied, Jeanmarie; St. George, Kirsten

    2012-01-01

    SUMMARY This report describes the development and pre-clinical testing of a new, random-access RNA sample preparation system (TruTip) for nasopharyngeal samples. The system is based on a monolithic, porous nucleic acid binding matrix embedded within an aerosol-resistant pipette tip and can be operated with single or multi-channel pipettors. Equivalent extraction efficiencies were obtained between automated QIAcube and manual TruTip methods at 106 gene copies influenza A per mL nasopharyngeal aspirate. Influenza A and B amended into nasopharyngeal swabs (in viral transport medium) were detected by real-time RT-PCR at approximately 745 and 370 gene copies per extraction, respectively. RNA extraction efficiency in nasopharyngeal swabs was also comparable to that obtained on an automated QIAcube instrument over a range of input concentrations; the correlation between threshold cycles (or nucleic acid recovery) for TruTip and QIAcube-purified RNA was R2 > 0.99. Preclinical testing of TruTip on blinded nasopharyngeal swab samples resulted in 98% detection accuracy relative to a clinically validated easyMAG extraction method. The physical properties of the TruTip binding matrix and ability to customize its shape and dimensions likewise make it amenable to automation and/or fluidic integration. PMID:22425698

  10. Automated Fluid Feature Extraction from Transient Simulations

    NASA Technical Reports Server (NTRS)

    Haimes, Robert; Lovely, David

    1999-01-01

    In the past, feature extraction and identification were interesting concepts, but not required to understand the underlying physics of a steady flow field. This is because the results of the more traditional tools like iso-surfaces, cuts and streamlines were more interactive and easily abstracted so they could be represented to the investigator. These tools worked and properly conveyed the collected information at the expense of much interaction. For unsteady flow-fields, the investigator does not have the luxury of spending time scanning only one "snap-shot" of the simulation. Automated assistance is required in pointing out areas of potential interest contained within the flow. This must not require a heavy compute burden (the visualization should not significantly slow down the solution procedure for co-processing environments like pV3). And methods must be developed to abstract the feature and display it in a manner that physically makes sense. The following is a list of the important physical phenomena found in transient (and steady-state) fluid flow: (1) Shocks, (2) Vortex cores, (3) Regions of recirculation, (4) Boundary layers, (5) Wakes. Three papers and an initial specification for the (The Fluid eXtraction tool kit) FX Programmer's guide were included. The papers, submitted to the AIAA Computational Fluid Dynamics Conference, are entitled : (1) Using Residence Time for the Extraction of Recirculation Regions, (2) Shock Detection from Computational Fluid Dynamics results and (3) On the Velocity Gradient Tensor and Fluid Feature Extraction.

  11. Automated Extraction of Secondary Flow Features

    NASA Technical Reports Server (NTRS)

    Dorney, Suzanne M.; Haimes, Robert

    2005-01-01

    The use of Computational Fluid Dynamics (CFD) has become standard practice in the design and development of the major components used for air and space propulsion. To aid in the post-processing and analysis phase of CFD many researchers now use automated feature extraction utilities. These tools can be used to detect the existence of such features as shocks, vortex cores and separation and re-attachment lines. The existence of secondary flow is another feature of significant importance to CFD engineers. Although the concept of secondary flow is relatively understood there is no commonly accepted mathematical definition for secondary flow. This paper will present a definition for secondary flow and one approach for automatically detecting and visualizing secondary flow.

  12. Automated Fluid Feature Extraction from Transient Simulations

    NASA Technical Reports Server (NTRS)

    Haimes, Robert

    2000-01-01

    In the past, feature extraction and identification were interesting concepts, but not required in understanding the physics of a steady flow field. This is because the results of the more traditional tools like iso-surfaces, cuts and streamlines, were more interactive and easily abstracted so they could be represented to the investigator. These tools worked and properly conveyed the collected information at the expense of a great deal of interaction. For unsteady flow-fields, the investigator does not have the luxury of spending time scanning only one 'snap-shot' of the simulation. Automated assistance is required in pointing out areas of potential interest contained within the flow. This must not require a heavy compute burden (the visualization should not significantly slow down the solution procedure for co-processing environments like pV3). And methods must be developed to abstract the feature and display it in a manner that physically makes sense.

  13. The RNA 3D Motif Atlas: Computational methods for extraction, organization and evaluation of RNA motifs.

    PubMed

    Parlea, Lorena G; Sweeney, Blake A; Hosseini-Asanjan, Maryam; Zirbel, Craig L; Leontis, Neocles B

    2016-07-01

    RNA 3D motifs occupy places in structured RNA molecules that correspond to the hairpin, internal and multi-helix junction "loops" of their secondary structure representations. As many as 40% of the nucleotides of an RNA molecule can belong to these structural elements, which are distinct from the regular double helical regions formed by contiguous AU, GC, and GU Watson-Crick basepairs. With the large number of atomic- or near atomic-resolution 3D structures appearing in a steady stream in the PDB/NDB structure databases, the automated identification, extraction, comparison, clustering and visualization of these structural elements presents an opportunity to enhance RNA science. Three broad applications are: (1) identification of modular, autonomous structural units for RNA nanotechnology, nanobiology and synthetic biology applications; (2) bioinformatic analysis to improve RNA 3D structure prediction from sequence; and (3) creation of searchable databases for exploring the binding specificities, structural flexibility, and dynamics of these RNA elements. In this contribution, we review methods developed for computational extraction of hairpin and internal loop motifs from a non-redundant set of high-quality RNA 3D structures. We provide a statistical summary of the extracted hairpin and internal loop motifs in the most recent version of the RNA 3D Motif Atlas. We also explore the reliability and accuracy of the extraction process by examining its performance in clustering recurrent motifs from homologous ribosomal RNA (rRNA) structures. We conclude with a summary of remaining challenges, especially with regard to extraction of multi-helix junction motifs. Copyright © 2016 Elsevier Inc. All rights reserved.

  14. An alternate method for DNA and RNA extraction from clotted blood.

    PubMed

    Zakaria, Z; Umi, S H; Mokhtar, S S; Mokhtar, U; Zaiharina, M Z; Aziz, A T A; Hoh, B P

    2013-02-04

    We developed an alternative method to extract DNA and RNA from clotted blood for genomic and molecular investigations. A combination of the TRIzol method and the QIAamp spin column were used to extract RNA from frozen clotted blood. Clotted blood was sonicated and then the QIAamp DNA Blood Mini Kit was used for DNA extraction. Extracted DNA and RNA were adequate for gene expression analysis and copy number variation (CNV) genotyping, respectively. The purity of the extracted RNA and DNA was in the range of 1.8-2.0, determined by absorbance ratios of A(260):A(280). Good DNA and RNA integrity were confirmed using gel electrophoresis and automated electrophoresis. The extracted DNA was suitable for qPCR and microarrays for CNV genotyping, while the extracted RNA was adequate for gene analysis using RT-qPCR.

  15. Automated Fluid Feature Extraction from Transient Simulations

    NASA Technical Reports Server (NTRS)

    Haimes, Robert

    1998-01-01

    In the past, feature extraction and identification were interesting concepts, but not required to understand the underlying physics of a steady flow field. This is because the results of the more traditional tools like iso-surfaces, cuts and streamlines were more interactive and easily abstracted so they could be represented to the investigator. These tools worked and properly conveyed the collected information at the expense of much interaction. For unsteady flow-fields, the investigator does not have the luxury of spending time scanning only one 'snap-shot' of the simulation. Automated assistance is required in pointing out areas of potential interest contained within the flow. This must not require a heavy compute burden (the visualization should not significantly slow down the solution procedure for co-processing environments like pV3). And methods must be developed to abstract the feature and display it in a manner that physically makes sense. The following is a list of the important physical phenomena found in transient (and steady-state) fluid flow: Shocks; Vortex ores; Regions of Recirculation; Boundary Layers; Wakes.

  16. RNA-protein binding kinetics in an automated microfluidic reactor.

    PubMed

    Ridgeway, William K; Seitaridou, Effrosyni; Phillips, Rob; Williamson, James R

    2009-11-01

    Microfluidic chips can automate biochemical assays on the nanoliter scale, which is of considerable utility for RNA-protein binding reactions that would otherwise require large quantities of proteins. Unfortunately, complex reactions involving multiple reactants cannot be prepared in current microfluidic mixer designs, nor is investigation of long-time scale reactions possible. Here, a microfluidic 'Riboreactor' has been designed and constructed to facilitate the study of kinetics of RNA-protein complex formation over long time scales. With computer automation, the reactor can prepare binding reactions from any combination of eight reagents, and is optimized to monitor long reaction times. By integrating a two-photon microscope into the microfluidic platform, 5-nl reactions can be observed for longer than 1000 s with single-molecule sensitivity and negligible photobleaching. Using the Riboreactor, RNA-protein binding reactions with a fragment of the bacterial 30S ribosome were prepared in a fully automated fashion and binding rates were consistent with rates obtained from conventional assays. The microfluidic chip successfully combines automation, low sample consumption, ultra-sensitive fluorescence detection and a high degree of reproducibility. The chip should be able to probe complex reaction networks describing the assembly of large multicomponent RNPs such as the ribosome.

  17. Automated feature extraction and classification from image sources

    USGS Publications Warehouse

    ,

    1995-01-01

    The U.S. Department of the Interior, U.S. Geological Survey (USGS), and Unisys Corporation have completed a cooperative research and development agreement (CRADA) to explore automated feature extraction and classification from image sources. The CRADA helped the USGS define the spectral and spatial resolution characteristics of airborne and satellite imaging sensors necessary to meet base cartographic and land use and land cover feature classification requirements and help develop future automated geographic and cartographic data production capabilities. The USGS is seeking a new commercial partner to continue automated feature extraction and classification research and development.

  18. Replacing β-mercaptoethanol in RNA extractions.

    PubMed

    Mommaerts, Kathleen; Sanchez, Ignacio; Betsou, Fay; Mathieson, William

    2015-06-15

    RNA extractions are potentially compromised in terms of both yield and quality by ribonucleases (RNases). The pungent and toxic reducing agent β-mercaptoethanol (β-ME), therefore, is commonly added to the biospecimen's lysis buffer to aid in RNase deactivation. Using different tissue types (liver tissue, kidney tissue, and cell pellets), extraction kits (RNeasy Mini Kit, Illustra RNA Spin Mini Kit, and PureLink Mini Kit), RNA quality assays (RNA integrity numbers [RINs] and quantitative real-time polymerase chain reaction [qRT-PCR]), yield assessments, and in vitro functional RNase assays (RNaseAlert Kit), we demonstrate that β-ME should be replaced by the less toxic dithiothreitol (DTT) alternative.

  19. Automated DNA extraction for large numbers of plant samples.

    PubMed

    Mehle, Nataša; Nikolić, Petra; Rupar, Matevž; Boben, Jana; Ravnikar, Maja; Dermastia, Marina

    2013-01-01

    The method described here is a rapid, total DNA extraction procedure applicable to a large number of plant samples requiring pathogen detection. The procedure combines a simple and quick homogenization step of crude extracts with DNA extraction based upon the binding of DNA to magnetic beads. DNA is purified in an automated process in which the magnetic beads are transferred through a series of washing buffers. The eluted DNA is suitable for efficient amplification in PCR reactions.

  20. [DNA extraction from bones and teeth using AutoMate Express forensic DNA extraction system].

    PubMed

    Gao, Lin-Lin; Xu, Nian-Lai; Xie, Wei; Ding, Shao-Cheng; Wang, Dong-Jing; Ma, Li-Qin; Li, You-Ying

    2013-04-01

    To explore a new method in order to extract DNA from bones and teeth automatically. Samples of 33 bones and 15 teeth were acquired by freeze-mill method and manual method, respectively. DNA materials were extracted and quantified from the triturated samples by AutoMate Express forensic DNA extraction system. DNA extraction from bones and teeth were completed in 3 hours using the AutoMate Express forensic DNA extraction system. There was no statistical difference between the two methods in the DNA concentration of bones. Both bones and teeth got the good STR typing by freeze-mill method, and the DNA concentration of teeth was higher than those by manual method. AutoMate Express forensic DNA extraction system is a new method to extract DNA from bones and teeth, which can be applied in forensic practice.

  1. Automating data extraction in systematic reviews: a systematic review.

    PubMed

    Jonnalagadda, Siddhartha R; Goyal, Pawan; Huffman, Mark D

    2015-06-15

    Automation of the parts of systematic review process, specifically the data extraction step, may be an important strategy to reduce the time necessary to complete a systematic review. However, the state of the science of automatically extracting data elements from full texts has not been well described. This paper performs a systematic review of published and unpublished methods to automate data extraction for systematic reviews. We systematically searched PubMed, IEEEXplore, and ACM Digital Library to identify potentially relevant articles. We included reports that met the following criteria: 1) methods or results section described what entities were or need to be extracted, and 2) at least one entity was automatically extracted with evaluation results that were presented for that entity. We also reviewed the citations from included reports. Out of a total of 1190 unique citations that met our search criteria, we found 26 published reports describing automatic extraction of at least one of more than 52 potential data elements used in systematic reviews. For 25 (48 %) of the data elements used in systematic reviews, there were attempts from various researchers to extract information automatically from the publication text. Out of these, 14 (27 %) data elements were completely extracted, but the highest number of data elements extracted automatically by a single study was 7. Most of the data elements were extracted with F-scores (a mean of sensitivity and positive predictive value) of over 70 %. We found no unified information extraction framework tailored to the systematic review process, and published reports focused on a limited (1-7) number of data elements. Biomedical natural language processing techniques have not been fully utilized to fully or even partially automate the data extraction step of systematic reviews.

  2. Efficient and scalable serial extraction of DNA and RNA from frozen tissue samples.

    PubMed

    Mathot, Lucy; Lindman, Monica; Sjöblom, Tobias

    2011-01-07

    Advances in cancer genomics have created a demand for scalable sample processing. We here present a process for serial extraction of nucleic acids from the same frozen tissue sample based on magnetic silica particles. The process is automation friendly with high recoveries of pure DNA and RNA suitable for analysis.

  3. Efficient oil palm total RNA extraction with a total RNA extraction kit.

    PubMed

    Habib, S H; Saud, H M; Kausar, H

    2014-04-03

    Oil palm tissues are rich in polyphenols, polysaccharides and secondary metabolites; these can co-precipitate with RNA, causing problems for downstream applications. We compared two different methods (one conventional and a kit-based method - Easy-Blue(TM) Total RNA Extraction Kit) to isolate total RNA from leaves, roots and shoot apical meristems of tissue culture derived truncated leaf syndrome somaclonal oil palm seedlings. The quality and quantity of total RNA were compared through spectrophotometry and formaldehyde gel electrophoresis. The specificity and applicability of the protocols were evaluated for downstream applications, including cDNA synthesis and RT-PCR analysis. We found that the conventional method gave higher yields of RNA but took longer, and it was contaminated with genomic DNA. This method required extra genomic DNA removal steps that further reduced the RNA yield. The kit-based method, on the other hand, produced good yields as well as well as good quality RNA, within a very short period of time from a small amount of starting material. Moreover, the RNA from the kit-based method was more suitable for synthesizing cDNA and RT-PCR amplification than the conventional method. Therefore, we conclude that the Easy-BlueTM Total RNA Extraction Kit method is suitable and superior for isolation of total RNA from oil palm leaf, root and shoot apical meristem.

  4. Extraction of high-quality RNA from human articular cartilage.

    PubMed

    Le Bleu, Heather K; Kamal, Fadia A; Kelly, Meghan; Ketz, John P; Zuscik, Michael J; Elbarbary, Reyad A

    2017-02-01

    Extracting high-quality RNA from articular cartilage is challenging due to low cellularity and high proteoglycan content. This problem hinders efficient application of RNA sequencing (RNA-seq) analysis in studying cartilage homeostasis. Here we developed a method that purifies high-quality RNA directly from cartilage. Our method optimized the collection and homogenization steps so as to minimize RNA degradation, and modified the conventional TRIzol protocol to enhance RNA purity. Cartilage RNA purified using our method has appropriate quality for RNA-seq experiments including an RNA integrity number of ∼8. Our method also proved efficient in extracting high-quality RNA from subchondral bone.

  5. Extrinsic Evaluation of Automated Information Extraction Programs

    DTIC Science & Technology

    2010-05-01

    promising IE tools; the former was developed by the Natural Language Processing ( NLP ) Group of the University of Sheffield and the latter by the Center...Human Intelligence (STEF HUMINT ) message set, and a Google message set. Extracted information can be visualized or formatted and stored as Resource...Since the project involved training Automap on only one of the three corpora, the STEF HUMINT message set, the delete list was aimed at removing

  6. Automated extraction of radiation dose information for CT examinations.

    PubMed

    Cook, Tessa S; Zimmerman, Stefan; Maidment, Andrew D A; Kim, Woojin; Boonn, William W

    2010-11-01

    Exposure to radiation as a result of medical imaging is currently in the spotlight, receiving attention from Congress as well as the lay press. Although scanner manufacturers are moving toward including effective dose information in the Digital Imaging and Communications in Medicine headers of imaging studies, there is a vast repository of retrospective CT data at every imaging center that stores dose information in an image-based dose sheet. As such, it is difficult for imaging centers to participate in the ACR's Dose Index Registry. The authors have designed an automated extraction system to query their PACS archive and parse CT examinations to extract the dose information stored in each dose sheet. First, an open-source optical character recognition program processes each dose sheet and converts the information to American Standard Code for Information Interchange (ASCII) text. Each text file is parsed, and radiation dose information is extracted and stored in a database which can be queried using an existing pathology and radiology enterprise search tool. Using this automated extraction pipeline, it is possible to perform dose analysis on the >800,000 CT examinations in the PACS archive and generate dose reports for all of these patients. It is also possible to more effectively educate technologists, radiologists, and referring physicians about exposure to radiation from CT by generating report cards for interpreted and performed studies. The automated extraction pipeline enables compliance with the ACR's reporting guidelines and greater awareness of radiation dose to patients, thus resulting in improved patient care and management.

  7. Automated vasculature extraction from placenta images

    NASA Astrophysics Data System (ADS)

    Almoussa, Nizar; Dutra, Brittany; Lampe, Bryce; Getreuer, Pascal; Wittman, Todd; Salafia, Carolyn; Vese, Luminita

    2011-03-01

    Recent research in perinatal pathology argues that analyzing properties of the placenta may reveal important information on how certain diseases progress. One important property is the structure of the placental blood vessels, which supply a fetus with all of its oxygen and nutrition. An essential step in the analysis of the vascular network pattern is the extraction of the blood vessels, which has only been done manually through a costly and time-consuming process. There is no existing method to automatically detect placental blood vessels; in addition, the large variation in the shape, color, and texture of the placenta makes it difficult to apply standard edge-detection algorithms. We describe a method to automatically detect and extract blood vessels from a given image by using image processing techniques and neural networks. We evaluate several local features for every pixel, in addition to a novel modification to an existing road detector. Pixels belonging to blood vessel regions have recognizable responses; hence, we use an artificial neural network to identify the pattern of blood vessels. A set of images where blood vessels are manually highlighted is used to train the network. We then apply the neural network to recognize blood vessels in new images. The network is effective in capturing the most prominent vascular structures of the placenta.

  8. Automated Image Registration Using Morphological Region of Interest Feature Extraction

    NASA Technical Reports Server (NTRS)

    Plaza, Antonio; LeMoigne, Jacqueline; Netanyahu, Nathan S.

    2005-01-01

    With the recent explosion in the amount of remotely sensed imagery and the corresponding interest in temporal change detection and modeling, image registration has become increasingly important as a necessary first step in the integration of multi-temporal and multi-sensor data for applications such as the analysis of seasonal and annual global climate changes, as well as land use/cover changes. The task of image registration can be divided into two major components: (1) the extraction of control points or features from images; and (2) the search among the extracted features for the matching pairs that represent the same feature in the images to be matched. Manual control feature extraction can be subjective and extremely time consuming, and often results in few usable points. Automated feature extraction is a solution to this problem, where desired target features are invariant, and represent evenly distributed landmarks such as edges, corners and line intersections. In this paper, we develop a novel automated registration approach based on the following steps. First, a mathematical morphology (MM)-based method is used to obtain a scale-orientation morphological profile at each image pixel. Next, a spectral dissimilarity metric such as the spectral information divergence is applied for automated extraction of landmark chips, followed by an initial approximate matching. This initial condition is then refined using a hierarchical robust feature matching (RFM) procedure. Experimental results reveal that the proposed registration technique offers a robust solution in the presence of seasonal changes and other interfering factors. Keywords-Automated image registration, multi-temporal imagery, mathematical morphology, robust feature matching.

  9. Automated sea floor extraction from underwater video

    NASA Astrophysics Data System (ADS)

    Kelly, Lauren; Rahmes, Mark; Stiver, James; McCluskey, Mike

    2016-05-01

    Ocean floor mapping using video is a method to simply and cost-effectively record large areas of the seafloor. Obtaining visual and elevation models has noteworthy applications in search and recovery missions. Hazards to navigation are abundant and pose a significant threat to the safety, effectiveness, and speed of naval operations and commercial vessels. This project's objective was to develop a workflow to automatically extract metadata from marine video and create image optical and elevation surface mosaics. Three developments made this possible. First, optical character recognition (OCR) by means of two-dimensional correlation, using a known character set, allowed for the capture of metadata from image files. Second, exploiting the image metadata (i.e., latitude, longitude, heading, camera angle, and depth readings) allowed for the determination of location and orientation of the image frame in mosaic. Image registration improved the accuracy of mosaicking. Finally, overlapping data allowed us to determine height information. A disparity map was created using the parallax from overlapping viewpoints of a given area and the relative height data was utilized to create a three-dimensional, textured elevation map.

  10. Automated Road Extraction from High Resolution Multispectral Imagery

    SciTech Connect

    Doucette, Peter J.; Agouris, Peggy; Stefanidis, Anthony

    2004-12-01

    Road networks represent a vital component of geospatial data sets in high demand, and thus contribute significantly to extraction labor costs. Multispectral imagery has only recently become widely available at high spatial resolutions, and modeling spectral content has received limited consideration for road extraction algorithms. This paper presents a methodology that exploits spectral content for fully automated road centerline extraction. Preliminary detection of road centerline pixel candidates is performed with Anti-parallel-edge Centerline Extraction (ACE). This is followed by constructing a road vector topology with a fuzzy grouping model that links nodes from a self-organized mapping of the ACE pixels. Following topology construction, a self-supervised road classification (SSRC) feedback loop is implemented to automate the process of training sample selection and refinement for a road class, as well deriving practical spectral definitions for non-road classes. SSRC demonstrates a potential to provide dramatic improvement in road extraction results by exploiting spectral content. Road centerline extraction results are presented for three 1m color-infrared suburban scenes, which show significant improvement following SSRC.

  11. Applications of the Automated SMAC Modal Parameter Extraction Package

    SciTech Connect

    MAYES,RANDALL L.; DORRELL,LARRY R.; KLENKE,SCOTT E.

    1999-10-29

    An algorithm known as SMAC (Synthesize Modes And Correlate), based on principles of modal filtering, has been in development for a few years. The new capabilities of the automated version are demonstrated on test data from a complex shell/payload system. Examples of extractions from impact and shaker data are shown. The automated algorithm extracts 30 to 50 modes in the bandwidth from each column of the frequency response function matrix. Examples of the synthesized Mode Indicator Functions (MIFs) compared with the actual MIFs show the accuracy of the technique. A data set for one input and 170 accelerometer outputs can typically be reduced in an hour. Application to a test with some complex modes is also demonstrated.

  12. Automated road extraction from aerial imagery by self-organization

    NASA Astrophysics Data System (ADS)

    Doucette, Peter J.

    To date, computer vision methods have largely focused on extraction from panchromatic imagery. Despite significant technological advances, road extraction algorithms have fallen short of satisfying rigorous production requirements. To that end, the objective of this thesis is to present a new approach for automating road detection from high-resolution multispectral imagery. This thesis considers three main research objectives: (1) development of a fully automated road extraction strategy in that interactive human supervision or input initializations are not required; (2) development of a globalized approach to road detection that is motivated by principles of self-organization; (3) meaningful exploitation of high-resolution multispectral imagery. Several new techniques are presented for fully automated road extraction from high-resolution imagery. The core algorithms implemented include (1) Anti-parallel edge Centerline Extractor (ACE), (2) Fuzzy Organization of Elongated Regions (FOrgER), and (3) Self-Organizing Road Finder (SORF). The ACE algorithm extends the idea of anti-parallel edge detection in a new approach that considers multi-layer images. The FOrgER algorithm is motivated by Gestalt grouping principles in perceptual organization. The FOrgER approach combines principles of self-organization with fuzzy inferencing to building road topology. Self-organization represents a learning paradigm that is neurobiologically motivated. Globalized analysis promotes lower sensitivity to fragmented information, and demonstrates robust capacity for handling scene clutter in high-resolution images. Finally, the SORF algorithm bridges concepts from ACE and FOrgER into a comprehensive and cooperative approach for fully automated road finding. By providing an exceptional breadth of input parameters, output metrics, modes of operation, and adaptability to various input, SORF is particularly well suited as an analytical research tool. Extraction results from the SORF

  13. Comparison of manual and automated nucleic acid isolation methods for HBV-DNA and HCV-RNA assays.

    PubMed

    Yagmur, Gulhan; Altun, Hatice Uludag; Gökahmetoglu, Selma; Basok, Ela

    2015-09-01

    In the diagnosis and monitoring of hepatitis B virus (HBV) and hepatitis C virus (HCV) infections, it is important to use methods that can provide rapid and reliable results. The present study aimed to compare the automated and manual extraction methods during the nucleic acid isolation phase for HBV-DNA and HCV-RNA assays. The study included 93 serum samples, 49 of which were for the HBV-DNA assay and 44 for the HCV-RNA assay. DNA and RNA isolation from the samples was performed manually with a "QIAmpMin Elute Kit" (Qiagen, Germany) and the automated isolation system, NucliSens easyMAG (BioMérieux, France). All the extraction products were amplified using the iCycler device (Bio-Rad, USA). With both methods, compliance was found in 21 (42.8%) samples in the HBV-DNA assay; nine (18.3%) samples had a higher amount of viral nucleic acid with the manual method, whereas 19 samples (38.7%) were found to have a higher amount of nucleic acid with the automated system. For the HCV-RNA assay, total compliance was found in 31 (70.4%) samples; 12 (27.2%) samples had a higher amount of viral nucleic acid with the manual method whereas one sample (2.2%) was found to have a higher amount of nucleic acid with the automated system. It was concluded that the NucliSens easyMAG automated isolation system can be used with confidence for nucleic acid extraction due to its higher sensitivity, providing results in a shorter time, and assured standardization.

  14. DNA, RNA, and protein extraction: the past and the present.

    PubMed

    Tan, Siun Chee; Yiap, Beow Chin

    2009-01-01

    Extraction of DNA, RNA, and protein is the basic method used in molecular biology. These biomolecules can be isolated from any biological material for subsequent downstream processes, analytical, or preparative purposes. In the past, the process of extraction and purification of nucleic acids used to be complicated, time-consuming, labor-intensive, and limited in terms of overall throughput. Currently, there are many specialized methods that can be used to extract pure biomolecules, such as solution-based and column-based protocols. Manual method has certainly come a long way over time with various commercial offerings which included complete kits containing most of the components needed to isolate nucleic acid, but most of them require repeated centrifugation steps, followed by removal of supernatants depending on the type of specimen and additional mechanical treatment. Automated systems designed for medium-to-large laboratories have grown in demand over recent years. It is an alternative to labor-intensive manual methods. The technology should allow a high throughput of samples; the yield, purity, reproducibility, and scalability of the biomolecules as well as the speed, accuracy, and reliability of the assay should be maximal, while minimizing the risk of cross-contamination.

  15. Automated DNA extraction platforms offer solutions to challenges of assessing microbial biofouling in oil production facilities

    PubMed Central

    2012-01-01

    The analysis of microbial assemblages in industrial, marine, and medical systems can inform decisions regarding quality control or mitigation. Modern molecular approaches to detect, characterize, and quantify microorganisms provide rapid and thorough measures unbiased by the need for cultivation. The requirement of timely extraction of high quality nucleic acids for molecular analysis is faced with specific challenges when used to study the influence of microorganisms on oil production. Production facilities are often ill equipped for nucleic acid extraction techniques, making the preservation and transportation of samples off-site a priority. As a potential solution, the possibility of extracting nucleic acids on-site using automated platforms was tested. The performance of two such platforms, the Fujifilm QuickGene-Mini80™ and Promega Maxwell®16 was compared to a widely used manual extraction kit, MOBIO PowerBiofilm™ DNA Isolation Kit, in terms of ease of operation, DNA quality, and microbial community composition. Three pipeline biofilm samples were chosen for these comparisons; two contained crude oil and corrosion products and the third transported seawater. Overall, the two more automated extraction platforms produced higher DNA yields than the manual approach. DNA quality was evaluated for amplification by quantitative PCR (qPCR) and end-point PCR to generate 454 pyrosequencing libraries for 16S rRNA microbial community analysis. Microbial community structure, as assessed by DGGE analysis and pyrosequencing, was comparable among the three extraction methods. Therefore, the use of automated extraction platforms should enhance the feasibility of rapidly evaluating microbial biofouling at remote locations or those with limited resources. PMID:23168231

  16. Improved Automated Seismic Event Extraction Using Machine Learning

    NASA Astrophysics Data System (ADS)

    Mackey, L.; Kleiner, A.; Jordan, M. I.

    2009-12-01

    Like many organizations engaged in seismic monitoring, the Preparatory Commission for the Comprehensive Test Ban Treaty Organization collects and processes seismic data from a large network of sensors. This data is continuously transmitted to a central data center, and bulletins of seismic events are automatically extracted. However, as for many such automated systems at present, the inaccuracy of this extraction necessitates substantial human analyst review effort. A significant opportunity for improvement thus lies in the fact that these systems currently fail to fully utilize the valuable repository of historical data provided by prior analyst reviews. In this work, we present the results of the application of machine learning approaches to several fundamental sub-tasks in seismic event extraction. These methods share as a common theme the use of historical analyst-reviewed bulletins as ground truth from which they extract relevant patterns to accomplish the desired goals. For instance, we demonstrate the effectiveness of classification and ranking methods for the identification of false events -- that is, those which will be invalidated and discarded by analysts -- in automated bulletins. We also show gains in the accuracy of seismic phase identification via the use of classification techniques to automatically assign seismic phase labels to station detections. Furthermore, we examine the potential of historical association data to inform the direct association of new signal detections with their corresponding seismic events. Empirical results are based upon parametric historical seismic detection and event data received from the Preparatory Commission for the Comprehensive Test Ban Treaty Organization.

  17. Automated genomic DNA extraction from saliva using the QIAxtractor.

    PubMed

    Keijzer, Henry; Endenburg, Silvia C; Smits, Marcel G; Koopmann, Miriam

    2010-05-01

    Venipuncture is an invasive procedure to obtain whole blood in order to obtain high quality and sufficient amounts of genomic DNA. Obtaining DNA from non-invasive sources is preferred by patients, medical doctors and researchers. Saliva collected with cotton swabs (Salivette) is increasingly being used to study chemical compounds, and it can also be a source of DNA. However, extracting DNA from Salivettes is very laborious and time consuming. Therefore, we developed a protocol for automated genomic DNA extraction from saliva collected in Salivette using the QIAxtractor. Saliva (0.1-2.0 mL) was collected by chewing on a Salivette for 1-2 min. A total of 70 samples, collected from healthy volunteers, were extracted with the QIAxtractor robot and a Qiagen DX reagent pack. Quantity and quality was assessed using UV spectrometry and real-time polymerase chain reaction (PCR) (substitution at position -729 in the CYP1A2 gene). The average DNA concentration from the saliva samples was 6.0 microg/mL (95% CI 5.4-6.6 microg/mL). In 100% of the saliva samples, PCR products were detected with an average cycle threshold of 23.1 (95% CI 22.6-23.6). DNA can be extracted in sufficient amounts from Salivette with a fully automated system with a short turnaround time. Real-time PCR can be performed with these samples.

  18. An automated approach for global identification of sRNA-encoding regions in RNA-Seq data from Mycobacterium tuberculosis.

    PubMed

    Wang, Ming; Fleming, Joy; Li, Zihui; Li, Chuanyou; Zhang, Hongtai; Xue, Yunxin; Chen, Maoshan; Zhang, Zongde; Zhang, Xian-En; Bi, Lijun

    2016-06-01

    Deep-sequencing of bacterial transcriptomes using RNA-Seq technology has made it possible to identify small non-coding RNAs, RNA molecules which regulate gene expression in response to changing environments, on a genome-wide scale in an ever-increasing range of prokaryotes. However, a simple and reliable automated method for identifying sRNA candidates in these large datasets is lacking. Here, after generating a transcriptome from an exponential phase culture of Mycobacterium tuberculosis H37Rv, we developed and validated an automated method for the genome-wide identification of sRNA candidate-containing regions within RNA-Seq datasets based on the analysis of the characteristics of reads coverage maps. We identified 192 novel candidate sRNA-encoding regions in intergenic regions and 664 RNA transcripts transcribed from regions antisense (as) to open reading frames (ORF), which bear the characteristics of asRNAs, and validated 28 of these novel sRNA-encoding regions by northern blotting. Our work has not only provided a simple automated method for genome-wide identification of candidate sRNA-encoding regions in RNA-Seq data, but has also uncovered many novel candidate sRNA-encoding regions in M. tuberculosis, reinforcing the view that the control of gene expression in bacteria is more complex than previously anticipated.

  19. ACME, a GIS tool for Automated Cirque Metric Extraction

    NASA Astrophysics Data System (ADS)

    Spagnolo, Matteo; Pellitero, Ramon; Barr, Iestyn D.; Ely, Jeremy C.; Pellicer, Xavier M.; Rea, Brice R.

    2017-02-01

    Regional scale studies of glacial cirque metrics provide key insights on the (palaeo) environment related to the formation of these erosional landforms. The growing availability of high resolution terrain models means that more glacial cirques can be identified and mapped in the future. However, the extraction of their metrics still largely relies on time consuming manual techniques or the combination of, more or less obsolete, GIS tools. In this paper, a newly coded toolbox is provided for the automated, and comparatively quick, extraction of 16 key glacial cirque metrics; including length, width, circularity, planar and 3D area, elevation, slope, aspect, plan closure and hypsometry. The set of tools, named ACME (Automated Cirque Metric Extraction), is coded in Python, runs in one of the most commonly used GIS packages (ArcGIS) and has a user friendly interface. A polygon layer of mapped cirques is required for all metrics, while a Digital Terrain Model and a point layer of cirque threshold midpoints are needed to run some of the tools. Results from ACME are comparable to those from other techniques and can be obtained rapidly, allowing large cirque datasets to be analysed and potentially important regional trends highlighted.

  20. Arduino-based automation of a DNA extraction system.

    PubMed

    Kim, Kyung-Won; Lee, Mi-So; Ryu, Mun-Ho; Kim, Jong-Won

    2015-01-01

    There have been many studies to detect infectious diseases with the molecular genetic method. This study presents an automation process for a DNA extraction system based on microfluidics and magnetic bead, which is part of a portable molecular genetic test system. This DNA extraction system consists of a cartridge with chambers, syringes, four linear stepper actuators, and a rotary stepper actuator. The actuators provide a sequence of steps in the DNA extraction process, such as transporting, mixing, and washing for the gene specimen, magnetic bead, and reagent solutions. The proposed automation system consists of a PC-based host application and an Arduino-based controller. The host application compiles a G code sequence file and interfaces with the controller to execute the compiled sequence. The controller executes stepper motor axis motion, time delay, and input-output manipulation. It drives the stepper motor with an open library, which provides a smooth linear acceleration profile. The controller also provides a homing sequence to establish the motor's reference position, and hard limit checking to prevent any over-travelling. The proposed system was implemented and its functionality was investigated, especially regarding positioning accuracy and velocity profile.

  1. Automated feature extraction for 3-dimensional point clouds

    NASA Astrophysics Data System (ADS)

    Magruder, Lori A.; Leigh, Holly W.; Soderlund, Alexander; Clymer, Bradley; Baer, Jessica; Neuenschwander, Amy L.

    2016-05-01

    Light detection and ranging (LIDAR) technology offers the capability to rapidly capture high-resolution, 3-dimensional surface data with centimeter-level accuracy for a large variety of applications. Due to the foliage-penetrating properties of LIDAR systems, these geospatial data sets can detect ground surfaces beneath trees, enabling the production of highfidelity bare earth elevation models. Precise characterization of the ground surface allows for identification of terrain and non-terrain points within the point cloud, and facilitates further discernment between natural and man-made objects based solely on structural aspects and relative neighboring parameterizations. A framework is presented here for automated extraction of natural and man-made features that does not rely on coincident ortho-imagery or point RGB attributes. The TEXAS (Terrain EXtraction And Segmentation) algorithm is used first to generate a bare earth surface from a lidar survey, which is then used to classify points as terrain or non-terrain. Further classifications are assigned at the point level by leveraging local spatial information. Similarly classed points are then clustered together into regions to identify individual features. Descriptions of the spatial attributes of each region are generated, resulting in the identification of individual tree locations, forest extents, building footprints, and 3-dimensional building shapes, among others. Results of the fully-automated feature extraction algorithm are then compared to ground truth to assess completeness and accuracy of the methodology.

  2. A simple automated instrument for DNA extraction in forensic casework.

    PubMed

    Montpetit, Shawn A; Fitch, Ian T; O'Donnell, Patrick T

    2005-05-01

    The Qiagen BioRobot EZ1 is a small, rapid, and reliable automated DNA extraction instrument capable of extracting DNA from up to six samples in as few as 20 min using magnetic bead technology. The San Diego Police Department Crime Laboratory has validated the BioRobot EZ1 for the DNA extraction of evidence and reference samples in forensic casework. The BioRobot EZ1 was evaluated for use on a variety of different evidence sample types including blood, saliva, and semen evidence. The performance of the BioRobot EZ1 with regard to DNA recovery and potential cross-contamination was also assessed. DNA yields obtained with the BioRobot EZ1 were comparable to those from organic extraction. The BioRobot EZ1 was effective at removing PCR inhibitors, which often co-purify with DNA in organic extractions. The incorporation of the BioRobot EZ1 into forensic casework has streamlined the DNA analysis process by reducing the need for labor-intensive phenol-chloroform extractions.

  3. Automated labeling of bibliographic data extracted from biomedical online journals

    NASA Astrophysics Data System (ADS)

    Kim, Jongwoo; Le, Daniel X.; Thoma, George R.

    2003-01-01

    A prototype system has been designed to automate the extraction of bibliographic data (e.g., article title, authors, abstract, affiliation and others) from online biomedical journals to populate the National Library of Medicine"s MEDLINE database. This paper describes a key module in this system: the labeling module that employs statistics and fuzzy rule-based algorithms to identify segmented zones in an article"s HTML pages as specific bibliographic data. Results from experiments conducted with 1,149 medical articles from forty-seven journal issues are presented.

  4. Comparison of commercial systems for extraction of nucleic acids from DNA/RNA respiratory pathogens.

    PubMed

    Yang, Genyan; Erdman, Dean E; Kodani, Maja; Kools, John; Bowen, Michael D; Fields, Barry S

    2011-01-01

    This study compared six automated nucleic acid extraction systems and one manual kit for their ability to recover nucleic acids from human nasal wash specimens spiked with five respiratory pathogens, representing Gram-positive bacteria (Streptococcus pyogenes), Gram-negative bacteria (Legionella pneumophila), DNA viruses (adenovirus), segmented RNA viruses (human influenza virus A), and non-segmented RNA viruses (respiratory syncytial virus). The robots and kit evaluated represent major commercially available methods that are capable of simultaneous extraction of DNA and RNA from respiratory specimens, and included platforms based on magnetic-bead technology (KingFisher mL, Biorobot EZ1, easyMAG, KingFisher Flex, and MagNA Pure Compact) or glass fiber filter technology (Biorobot MDX and the manual kit Allprep). All methods yielded extracts free of cross-contamination and RT-PCR inhibition. All automated systems recovered L. pneumophila and adenovirus DNA equivalently. However, the MagNA Pure protocol demonstrated more than 4-fold higher DNA recovery from the S. pyogenes than other methods. The KingFisher mL and easyMAG protocols provided 1- to 3-log wider linearity and extracted 3- to 4-fold more RNA from the human influenza virus and respiratory syncytial virus. These findings suggest that systems differed in nucleic acid recovery, reproducibility, and linearity in a pathogen specific manner. Published by Elsevier B.V.

  5. Automated Feature Extraction of Foredune Morphology from Terrestrial Lidar Data

    NASA Astrophysics Data System (ADS)

    Spore, N.; Brodie, K. L.; Swann, C.

    2014-12-01

    Foredune morphology is often described in storm impact prediction models using the elevation of the dune crest and dune toe and compared with maximum runup elevations to categorize the storm impact and predicted responses. However, these parameters do not account for other foredune features that may make them more or less erodible, such as alongshore variations in morphology, vegetation coverage, or compaction. The goal of this work is to identify other descriptive features that can be extracted from terrestrial lidar data that may affect the rate of dune erosion under wave attack. Daily, mobile-terrestrial lidar surveys were conducted during a 6-day nor'easter (Hs = 4 m in 6 m water depth) along 20km of coastline near Duck, North Carolina which encompassed a variety of foredune forms in close proximity to each other. This abstract will focus on the tools developed for the automated extraction of the morphological features from terrestrial lidar data, while the response of the dune will be presented by Brodie and Spore as an accompanying abstract. Raw point cloud data can be dense and is often under-utilized due to time and personnel constraints required for analysis, since many algorithms are not fully automated. In our approach, the point cloud is first projected into a local coordinate system aligned with the coastline, and then bare earth points are interpolated onto a rectilinear 0.5 m grid creating a high resolution digital elevation model. The surface is analyzed by identifying features along each cross-shore transect. Surface curvature is used to identify the position of the dune toe, and then beach and berm morphology is extracted shoreward of the dune toe, and foredune morphology is extracted landward of the dune toe. Changes in, and magnitudes of, cross-shore slope, curvature, and surface roughness are used to describe the foredune face and each cross-shore transect is then classified using its pre-storm morphology for storm-response analysis.

  6. Automated blood vessel extraction using local features on retinal images

    NASA Astrophysics Data System (ADS)

    Hatanaka, Yuji; Samo, Kazuki; Tajima, Mikiya; Ogohara, Kazunori; Muramatsu, Chisako; Okumura, Susumu; Fujita, Hiroshi

    2016-03-01

    An automated blood vessel extraction using high-order local autocorrelation (HLAC) on retinal images is presented. Although many blood vessel extraction methods based on contrast have been proposed, a technique based on the relation of neighbor pixels has not been published. HLAC features are shift-invariant; therefore, we applied HLAC features to retinal images. However, HLAC features are weak to turned image, thus a method was improved by the addition of HLAC features to a polar transformed image. The blood vessels were classified using an artificial neural network (ANN) with HLAC features using 105 mask patterns as input. To improve performance, the second ANN (ANN2) was constructed by using the green component of the color retinal image and the four output values of ANN, Gabor filter, double-ring filter and black-top-hat transformation. The retinal images used in this study were obtained from the "Digital Retinal Images for Vessel Extraction" (DRIVE) database. The ANN using HLAC output apparent white values in the blood vessel regions and could also extract blood vessels with low contrast. The outputs were evaluated using the area under the curve (AUC) based on receiver operating characteristics (ROC) analysis. The AUC of ANN2 was 0.960 as a result of our study. The result can be used for the quantitative analysis of the blood vessels.

  7. Extraction of high-quality RNA from rubber tree leaves.

    PubMed

    Deng, Liu-Hong; Luo, Ming-Wu; Zhang, Chun-Fa; Zeng, Hui-Cai

    2012-01-01

    A specific technique capable of producing high-quality RNA for rapid amplification of cDNA ends (RACE) was established for challenging tissues: leaves of the rubber tree. Total RNA was extracted by cetyltrimethylammonium bromide (CTAB)-LiCl combined with TRIzol reagent. The isolated RNA was highly intact. With RNA as template, full-length cDNA was obtained (NCBI, AY461413) by RACE.

  8. Automated extraction of knowledge for model-based diagnostics

    NASA Technical Reports Server (NTRS)

    Gonzalez, Avelino J.; Myler, Harley R.; Towhidnejad, Massood; Mckenzie, Frederic D.; Kladke, Robin R.

    1990-01-01

    The concept of accessing computer aided design (CAD) design databases and extracting a process model automatically is investigated as a possible source for the generation of knowledge bases for model-based reasoning systems. The resulting system, referred to as automated knowledge generation (AKG), uses an object-oriented programming structure and constraint techniques as well as internal database of component descriptions to generate a frame-based structure that describes the model. The procedure has been designed to be general enough to be easily coupled to CAD systems that feature a database capable of providing label and connectivity data from the drawn system. The AKG system is capable of defining knowledge bases in formats required by various model-based reasoning tools.

  9. Automated and assisted RNA resonance assignment using NMR chemical shift statistics.

    PubMed

    Aeschbacher, Thomas; Schmidt, Elena; Blatter, Markus; Maris, Christophe; Duss, Olivier; Allain, Frédéric H-T; Güntert, Peter; Schubert, Mario

    2013-10-01

    The three-dimensional structure determination of RNAs by NMR spectroscopy relies on chemical shift assignment, which still constitutes a bottleneck. In order to develop more efficient assignment strategies, we analysed relationships between sequence and (1)H and (13)C chemical shifts. Statistics of resonances from regularly Watson-Crick base-paired RNA revealed highly characteristic chemical shift clusters. We developed two approaches using these statistics for chemical shift assignment of double-stranded RNA (dsRNA): a manual approach that yields starting points for resonance assignment and simplifies decision trees and an automated approach based on the recently introduced automated resonance assignment algorithm FLYA. Both strategies require only unlabeled RNAs and three 2D spectra for assigning the H2/C2, H5/C5, H6/C6, H8/C8 and H1'/C1' chemical shifts. The manual approach proved to be efficient and robust when applied to the experimental data of RNAs with a size between 20 nt and 42 nt. The more advanced automated assignment approach was successfully applied to four stem-loop RNAs and a 42 nt siRNA, assigning 92-100% of the resonances from dsRNA regions correctly. This is the first automated approach for chemical shift assignment of non-exchangeable protons of RNA and their corresponding (13)C resonances, which provides an important step toward automated structure determination of RNAs.

  10. Automated and assisted RNA resonance assignment using NMR chemical shift statistics

    PubMed Central

    Aeschbacher, Thomas; Schmidt, Elena; Blatter, Markus; Maris, Christophe; Duss, Olivier; Allain, Frédéric H.-T.; Güntert, Peter; Schubert, Mario

    2013-01-01

    The three-dimensional structure determination of RNAs by NMR spectroscopy relies on chemical shift assignment, which still constitutes a bottleneck. In order to develop more efficient assignment strategies, we analysed relationships between sequence and 1H and 13C chemical shifts. Statistics of resonances from regularly Watson–Crick base-paired RNA revealed highly characteristic chemical shift clusters. We developed two approaches using these statistics for chemical shift assignment of double-stranded RNA (dsRNA): a manual approach that yields starting points for resonance assignment and simplifies decision trees and an automated approach based on the recently introduced automated resonance assignment algorithm FLYA. Both strategies require only unlabeled RNAs and three 2D spectra for assigning the H2/C2, H5/C5, H6/C6, H8/C8 and H1′/C1′ chemical shifts. The manual approach proved to be efficient and robust when applied to the experimental data of RNAs with a size between 20 nt and 42 nt. The more advanced automated assignment approach was successfully applied to four stem-loop RNAs and a 42 nt siRNA, assigning 92–100% of the resonances from dsRNA regions correctly. This is the first automated approach for chemical shift assignment of non-exchangeable protons of RNA and their corresponding 13C resonances, which provides an important step toward automated structure determination of RNAs. PMID:23921634

  11. An automated procedure for covariation-based detection of RNA structure

    SciTech Connect

    Winker, S.; Overbeek, R.; Woese, C.R.; Olsen, G.J.; Pfluger, N.

    1989-12-01

    This paper summarizes our investigations into the computational detection of secondary and tertiary structure of ribosomal RNA. We have developed a new automated procedure that not only identifies potential bondings of secondary and tertiary structure, but also provides the covariation evidence that supports the proposed bondings, and any counter-evidence that can be detected in the known sequences. A small number of previously unknown bondings have been detected in individual RNA molecules (16S rRNA and 7S RNA) through the use of our automated procedure. Currently, we are systematically studying mitochondrial rRNA. Our goal is to detect tertiary structure within 16S rRNA and quaternary structure between 16S and 23S rRNA. Our ultimate hope is that automated covariation analysis will contribute significantly to a refined picture of ribosome structure. Our colleagues in biology have begun experiments to test certain hypotheses suggested by an examination of our program's output. These experiments involve sequencing key portions of the 23S ribosomal RNA for species in which the known 16S ribosomal RNA exhibits variation (from the dominant pattern) at the site of a proposed bonding. The hope is that the 23S ribosomal RNA of these species will exhibit corresponding complementary variation or generalized covariation. 24 refs.

  12. Semi-automated extraction of microbial DNA from feces for qPCR and phylogenetic microarray analysis.

    PubMed

    Nylund, Lotta; Heilig, Hans G H J; Salminen, Seppo; de Vos, Willem M; Satokari, Reetta

    2010-11-01

    The human gastrointestinal tract (GI-tract) harbors a complex microbial ecosystem, largely composed of so far uncultured species, which can be detected only by using techniques such as PCR and by different hybridization techniques including phylogenetic microarrays. Manual DNA extraction from feces is laborious and is one of the bottlenecks holding up the application of microarray and other DNA-based techniques in large cohort studies. In order to enhance the DNA extraction step we combined mechanical disruption of microbial cells by repeated bead-beating (RBB) with two automated DNA extraction methods, KingFisher with InviMag Stool DNA kit (KF) and NucliSENS easyMAG (NeM). The semi-automated DNA extraction methods, RBB combined with either KF or NeM, were compared to the manual extraction method currently considered the most suited method for fecal DNA extraction by assessing the yield of 16S rRNA gene copies by qPCR and total microbiota composition by the HITChip, a phylogenetic microarray. Parallel DNA extractions from infant fecal samples by using the three methods showed that the KF and manual methods gave comparable yields of 16S rRNA gene copies as assessed by qPCR, whereas NeM showed a significantly lower yield. All three methods showed highly similar microbiota profiles in HITChip. Both KF and NeM were found to be suitable methods for DNA extraction from fecal samples after the mechanical disruption of microbial cells by bead-beating. The semi-automated methods could be performed in half of the time required for the manual protocol, while being comparable to the manual method in terms of reagent costs. Copyright © 2010 Elsevier B.V. All rights reserved.

  13. Extraction of polychlorinated biphenyls from soils by automated focused microwave-assisted Soxhlet extraction.

    PubMed

    Luque-García, J L; de Castro, Luque

    2003-05-23

    The application of a new focused microwave-assisted Soxhlet extractor for the extraction of polychlorinated biphenyls from differently aged soils is here presented. The new extractor overcomes the disadvantages of previous devices based on the same principle and enables a fully automated extraction of two samples simultaneously. The variables affecting the extraction step (namely, power of irradiation, irradiation time, extractant volume, extractant composition and number of extraction cycles) have been optimized using experimental design methodology. The optimized method has also been applied to a certified reference material (CRM910-050 "real" contaminated soil) for quality assurance validation. Quantification of the target compounds has been performed by GC with ion-trap MS. The mass spectrometer was operated in the electron-ionization mode, with selected-ion monitoring at m/z 152, 186, 292, 326 and 498. The results obtained have demonstrated that this approach is as efficient as conventional Soxhlet but with a drastic reduction of both extraction time (70 min vs. 24 h for the "real" contaminated soil) and organic solvent disposal, as 75-80% of the extractant is recycled.

  14. Automated Dsm Extraction from Uav Images and Performance Analysis

    NASA Astrophysics Data System (ADS)

    Rhee, S.; Kim, T.

    2015-08-01

    As technology evolves, unmanned aerial vehicles (UAVs) imagery is being used from simple applications such as image acquisition to complicated applications such as 3D spatial information extraction. Spatial information is usually provided in the form of a DSM or point cloud. It is important to generate very dense tie points automatically from stereo images. In this paper, we tried to apply stereo image-based matching technique developed for satellite/aerial images to UAV images, propose processing steps for automated DSM generation and to analyse the possibility of DSM generation. For DSM generation from UAV images, firstly, exterior orientation parameters (EOPs) for each dataset were adjusted. Secondly, optimum matching pairs were determined. Thirdly, stereo image matching was performed with each pair. Developed matching algorithm is based on grey-level correlation on pixels applied along epipolar lines. Finally, the extracted match results were united with one result and the final DSM was made. Generated DSM was compared with a reference DSM from Lidar. Overall accuracy was 1.5 m in NMAD. However, several problems have to be solved in future, including obtaining precise EOPs, handling occlusion and image blurring problems. More effective interpolation technique needs to be developed in the future.

  15. Automated Extraction of Substance Use Information from Clinical Texts.

    PubMed

    Wang, Yan; Chen, Elizabeth S; Pakhomov, Serguei; Arsoniadis, Elliot; Carter, Elizabeth W; Lindemann, Elizabeth; Sarkar, Indra Neil; Melton, Genevieve B

    2015-01-01

    Within clinical discourse, social history (SH) includes important information about substance use (alcohol, drug, and nicotine use) as key risk factors for disease, disability, and mortality. In this study, we developed and evaluated a natural language processing (NLP) system for automated detection of substance use statements and extraction of substance use attributes (e.g., temporal and status) based on Stanford Typed Dependencies. The developed NLP system leveraged linguistic resources and domain knowledge from a multi-site social history study, Propbank and the MiPACQ corpus. The system attained F-scores of 89.8, 84.6 and 89.4 respectively for alcohol, drug, and nicotine use statement detection, as well as average F-scores of 82.1, 90.3, 80.8, 88.7, 96.6, and 74.5 respectively for extraction of attributes. Our results suggest that NLP systems can achieve good performance when augmented with linguistic resources and domain knowledge when applied to a wide breadth of substance use free text clinical notes.

  16. Extraction of Bacterial RNA from Soil: Challenges and Solutions

    PubMed Central

    Wang, Yong; Hayatsu, Masahito; Fujii, Takeshi

    2012-01-01

    Detection of bacterial gene expression in soil emerged in the early 1990s and provided information on bacterial responses in their original soil environments. As a key procedure in the detection, extraction of bacterial RNA from soil has attracted much interest, and many methods of soil RNA extraction have been reported in the past 20 years. In addition to various RT-PCR-based technologies, new technologies for gene expression analysis, such as microarrays and high-throughput sequencing technologies, have recently been applied to examine bacterial gene expression in soil. These technologies are driving improvements in RNA extraction protocols. In this mini-review, progress in the extraction of bacterial RNA from soil is summarized with emphasis on the major difficulties in the development of methodologies and corresponding strategies to overcome them. PMID:22791042

  17. Automated concept and relationship extraction for the semi-automated ontology management (SEAM) system.

    PubMed

    Doing-Harris, Kristina; Livnat, Yarden; Meystre, Stephane

    2015-01-01

    We develop medical-specialty specific ontologies that contain the settled science and common term usage. We leverage current practices in information and relationship extraction to streamline the ontology development process. Our system combines different text types with information and relationship extraction techniques in a low overhead modifiable system. Our SEmi-Automated ontology Maintenance (SEAM) system features a natural language processing pipeline for information extraction. Synonym and hierarchical groups are identified using corpus-based semantics and lexico-syntactic patterns. The semantic vectors we use are term frequency by inverse document frequency and context vectors. Clinical documents contain the terms we want in an ontology. They also contain idiosyncratic usage and are unlikely to contain the linguistic constructs associated with synonym and hierarchy identification. By including both clinical and biomedical texts, SEAM can recommend terms from those appearing in both document types. The set of recommended terms is then used to filter the synonyms and hierarchical relationships extracted from the biomedical corpus. We demonstrate the generality of the system across three use cases: ontologies for acute changes in mental status, Medically Unexplained Syndromes, and echocardiogram summary statements. Across the three uses cases, we held the number of recommended terms relatively constant by changing SEAM's parameters. Experts seem to find more than 300 recommended terms to be overwhelming. The approval rate of recommended terms increased as the number and specificity of clinical documents in the corpus increased. It was 60% when there were 199 clinical documents that were not specific to the ontology domain and 90% when there were 2879 documents very specific to the target domain. We found that fewer than 100 recommended synonym groups were also preferred. Approval rates for synonym recommendations remained low varying from 43% to 25% as the

  18. A COMPARISON OF AUTOMATED AND TRADITIONAL METHODS FOR THE EXTRACTION OF ARSENICALS FROM FISH

    EPA Science Inventory

    An automated extractor employing accelerated solvent extraction (ASE) has been compared with a traditional sonication method of extraction for the extraction of arsenicals from fish tissue. Four different species of fish and a standard reference material, DORM-2, were subjected t...

  19. AUTOMATED SOLID PHASE EXTRACTION GC/MS FOR ANALYSIS OF SEMIVOLATILES IN WATER AND SEDIMENTS

    EPA Science Inventory

    Data is presented on the development of a new automated system combining solid phase extraction (SPE) with GC/MS spectrometry for the single-run analysis of water samples containing a broad range of organic compounds. The system uses commercially available automated in-line sampl...

  20. AUTOMATED SOLID PHASE EXTRACTION GC/MS FOR ANALYSIS OF SEMIVOLATILES IN WATER AND SEDIMENTS

    EPA Science Inventory

    Data is presented on the development of a new automated system combining solid phase extraction (SPE) with GC/MS spectrometry for the single-run analysis of water samples containing a broad range of organic compounds. The system uses commercially available automated in-line sampl...

  1. Automated discovery of active motifs in multiple RNA secondary structures

    SciTech Connect

    Wang, J.T.L.; Chang, Chia-Yo; Shapiro, B.A.

    1996-12-31

    In this paper we present a method for discovering approximately common motifs (also known as active motifs) in multiple RNA secondary structures. The secondary structures can be represented as ordered trees (i.e., the order among siblings matters). Motifs in these trees are connected subgraphs that can differ in both substitutions and deletions/insertions. The proposed method consists of two steps: (1) find candidate motifs in a small sample of the secondary structures; (2) search all of the secondary structures to determine how frequently these motifs occur (within the allowed approximation) in the secondary structures. To reduce the running time, we develop two optimization heuristics based on sampling and pattern matching techniques. Experimental results obtained by running these algorithms on both generated data and RNA secondary structures show the good performance of the algorithms. To demonstrate the utility of our algorithms, we discuss their applications to conducting the phylogenetic study of RNA sequences obtained from GenBank.

  2. PRI-Modeler: extracting RNA structural elements from PDB files of protein-RNA complexes.

    PubMed

    Han, Kyungsook; Nepal, Chirag

    2007-05-01

    A complete understanding of protein and RNA structures and their interactions is important for determining the binding sites in protein-RNA complexes. Computational approaches exist for identifying secondary structural elements in proteins from atomic coordinates. However, similar methods have not been developed for RNA, due in part to the very limited structural data so far available. We have developed a set of algorithms for extracting and visualizing secondary and tertiary structures of RNA and for analyzing protein-RNA complexes. These algorithms have been implemented in a web-based program called PRI-Modeler (protein-RNA interaction modeler). Given one or more protein data bank files of protein-RNA complexes, PRI-Modeler analyzes the conformation of the RNA, calculates the hydrogen bond (H bond) and van der Waals interactions between amino acids and nucleotides, extracts secondary and tertiary RNA structure elements, and identifies the patterns of interactions between the proteins and RNAs. This paper presents PRI-Modeler and its application to the hydrogen bond and van der Waals interactions in the most representative set of protein-RNA complexes. The analysis reveals several interesting interaction patterns at various levels. The information provided by PRI-Modeler should prove useful for determining the binding sites in protein-RNA complexes. PRI-Modeler is accessible at http://wilab.inha.ac.kr/primodeler/, and supplementary materials are available in the analysis results section at http://wilab.inha.ac.kr/primodeler/.

  3. Automated identification of reference genes based on RNA-seq data.

    PubMed

    Carmona, Rosario; Arroyo, Macarena; Jiménez-Quesada, María José; Seoane, Pedro; Zafra, Adoración; Larrosa, Rafael; Alché, Juan de Dios; Claros, M Gonzalo

    2017-08-18

    Gene expression analyses demand appropriate reference genes (RGs) for normalization, in order to obtain reliable assessments. Ideally, RG expression levels should remain constant in all cells, tissues or experimental conditions under study. Housekeeping genes traditionally fulfilled this requirement, but they have been reported to be less invariant than expected; therefore, RGs should be tested and validated for every particular situation. Microarray data have been used to propose new RGs, but only a limited set of model species and conditions are available; on the contrary, RNA-seq experiments are more and more frequent and constitute a new source of candidate RGs. An automated workflow based on mapped NGS reads has been constructed to obtain highly and invariantly expressed RGs based on a normalized expression in reads per mapped million and the coefficient of variation. This workflow has been tested with Roche/454 reads from reproductive tissues of olive tree (Olea europaea L.), as well as with Illumina paired-end reads from two different accessions of Arabidopsis thaliana and three different human cancers (prostate, small-cell cancer lung and lung adenocarcinoma). Candidate RGs have been proposed for each species and many of them have been previously reported as RGs in literature. Experimental validation of significant RGs in olive tree is provided to support the algorithm. Regardless sequencing technology, number of replicates, and library sizes, when RNA-seq experiments are designed and performed, the same datasets can be analyzed with our workflow to extract suitable RGs for subsequent PCR validation. Moreover, different subset of experimental conditions can provide different suitable RGs.

  4. Use of conventional bioanalytical devices to automate DBS extractions in liquid-handling dispensing tips.

    PubMed

    Johnson, Casey Jl; Christianson, Chad D; Sheaff, Chrystal N; Laine, Derek F; Zimmer, Jennifer Sd; Needham, Shane R

    2011-10-01

    Conventional liquid-handling devices were employed, along with an improved punching device, to semi-automate dried blood spot (DBS) extraction of alprazolam, α-hydroxyalprazolam and midazolam from human whole blood. Liquid-handling devices were used to add internal standard to the DBS cards and to extract the analytes from the DBS, in order to be analyzed by HPLC-MS/MS. The technique was shown to be accurate (±12.0%) and precise (10.3%) across the dynamic range of the assay. The semi-automated extraction reduced sample preparation time by more than 50% when compared with more conventional DBS manual extraction methods.

  5. Evaluation of automated and manual commercial DNA extraction methods for recovery of Brucella DNA from suspensions and spiked swabs.

    PubMed

    Dauphin, Leslie A; Hutchins, Rebecca J; Bost, Liberty A; Bowen, Michael D

    2009-12-01

    This study evaluated automated and manual commercial DNA extraction methods for their ability to recover DNA from Brucella species in phosphate-buffered saline (PBS) suspension and from spiked swab specimens. Six extraction methods, representing several of the methodologies which are commercially available for DNA extraction, as well as representing various throughput capacities, were evaluated: the MagNA Pure Compact and the MagNA Pure LC instruments, the IT 1-2-3 DNA sample purification kit, the MasterPure Complete DNA and RNA purification kit, the QIAamp DNA blood mini kit, and the UltraClean microbial DNA isolation kit. These six extraction methods were performed upon three pathogenic Brucella species: B. abortus, B. melitensis, and B. suis. Viability testing of the DNA extracts indicated that all six extraction methods were efficient at inactivating virulent Brucella spp. Real-time PCR analysis using Brucella genus- and species-specific TaqMan assays revealed that use of the MasterPure kit resulted in superior levels of detection from bacterial suspensions, while the MasterPure kit and MagNA Pure Compact performed equally well for extraction of spiked swab samples. This study demonstrated that DNA extraction methodologies differ in their ability to recover Brucella DNA from PBS bacterial suspensions and from swab specimens and, thus, that the extraction method used for a given type of sample matrix can influence the sensitivity of real-time PCR assays for Brucella.

  6. Purification of RNA by SDS solubilization and phenol extraction.

    PubMed

    Rio, Donald C; Ares, Manuel; Hannon, Gregory J; Nilsen, Timothy W

    2010-06-01

    This protocol describes a method for RNA purification by sodium dodecyl sulfate (SDS) solubilization and phenol extraction. It is of wide utility and is used routinely to deproteinize RNAs in biological material that has been solubilized in SDS, an ionic detergent that dissolves membranes, disrupts protein-nucleic acid interactions, and inactivates ribonucleases. Once solubilized, addition of phenol or phenol:chloroform:isoamyl alcohol (PCA) completely denatures the protein, and it becomes insoluble in aqueous solution. PCA extraction is the method of choice for preparing cytoplasmic RNA from tissue culture cells or in any other situation (e.g., enzyme reactions) where solubilization in SDS is easily achievable.

  7. Lab-on-a-chip mRNA purification and reverse transcription via a solid-phase gene extraction technique.

    PubMed

    Nestorova, Gergana G; Hasenstein, Karl; Nguyen, Nam; DeCoster, Mark A; Crews, Niel D

    2017-03-14

    Extraction and purification of high quality RNA is a crucial initial step required for a variety of genomic assays. We report a solid phase gene extraction (SPGE) method for automated extraction, purification and reverse transcription of mRNA in a microfluidic device. This is performed using a 130 μm diameter stainless steel needle that is amino-linked to dT(15) oligonucleotides for selective hybridization of mRNA. By inserting this probe into the biological sample for only 30 seconds, mRNA is captured with high selectivity and a yield greater than 10 pg per mm of probe length. The probe is then inserted into a lab-on-a-chip device, where the bound poly-adenylated RNA is thermally released and immediately reverse transcribed for subsequent PCR amplification. The insertion of the probe into the microfluidic device is straightforward: the microchannel is formed with an elastomer (PDMS) that, when punctured, will seal around the probe. The specificity and RNA loading capacity of the probes were evaluated using conventional qPCR. This procedure was successfully used to extract, purify, and transcribe mRNA from rat glioblastoma cell spheroids in less than seven minutes. Analysis of the product confirmed that the SPGE technique selectively captures and inherently purifies high-quality mRNA directly from biological material with no need for additional pre-processing steps. Integrating this elegant sample preparation method into a complete lab-on-a-chip system will substantially enhance the speed and automation of mRNA assays for research and clinical diagnostics.

  8. RNA Contaminates Glycosaminoglycans Extracted from Cells and Tissues.

    PubMed

    van Gemst, Jasper J; Loeven, Markus A; de Graaf, Mark J J; Berden, Jo H M; Rabelink, Ton J; Smit, Cornelis H; van der Vlag, Johan

    2016-01-01

    Glycosaminoglycans (GAGs) are linear negatively charged polysaccharides and important components of extracellular matrices and cell surface glycan layers such as the endothelial glycocalyx. The GAG family includes sulfated heparin, heparan sulfate (HS), dermatan sulfate (DS), chondroitin sulfate (CS), keratan sulfate, and non-sulfated hyaluronan. Because relative expression of GAGs is dependent on cell-type and niche, isolating GAGs from cell cultures and tissues may provide insight into cell- and tissue-specific GAG structure and functions. In our objective to obtain structural information about the GAGs expressed on a specialized mouse glomerular endothelial cell culture (mGEnC-1) we adapted a recently published GAG isolation protocol, based on cell lysis, proteinase K and DNase I digestion. Analysis of the GAGs contributing to the mGEnC-1 glycocalyx indicated a large HS and a minor CS content on barium acetate gel. However, isolated GAGs appeared resistant to enzymatic digestion by heparinases. We found that these GAG extracts were heavily contaminated with RNA, which co-migrated with HS in barium acetate gel electrophoresis and interfered with 1,9-dimethylmethylene blue (DMMB) assays, resulting in an overestimation of GAG yields. We hypothesized that RNA may be contaminating GAG extracts from other cell cultures and possibly tissue, and therefore investigated potential RNA contaminations in GAG extracts from two additional cell lines, human umbilical vein endothelial cells and retinal pigmental epithelial cells, and mouse kidney, liver, spleen and heart tissue. GAG extracts from all examined cell lines and tissues contained varying amounts of contaminating RNA, which interfered with GAG quantification using DMMB assays and characterization of GAGs by barium acetate gel electrophoresis. We therefore recommend routinely evaluating the RNA content of GAG extracts and propose a robust protocol for GAG isolation that includes an RNA digestion step.

  9. RNA Contaminates Glycosaminoglycans Extracted from Cells and Tissues

    PubMed Central

    de Graaf, Mark J. J.; Berden, Jo H. M.; Rabelink, Ton J.; Smit, Cornelis H.

    2016-01-01

    Glycosaminoglycans (GAGs) are linear negatively charged polysaccharides and important components of extracellular matrices and cell surface glycan layers such as the endothelial glycocalyx. The GAG family includes sulfated heparin, heparan sulfate (HS), dermatan sulfate (DS), chondroitin sulfate (CS), keratan sulfate, and non-sulfated hyaluronan. Because relative expression of GAGs is dependent on cell-type and niche, isolating GAGs from cell cultures and tissues may provide insight into cell- and tissue-specific GAG structure and functions. In our objective to obtain structural information about the GAGs expressed on a specialized mouse glomerular endothelial cell culture (mGEnC-1) we adapted a recently published GAG isolation protocol, based on cell lysis, proteinase K and DNase I digestion. Analysis of the GAGs contributing to the mGEnC-1 glycocalyx indicated a large HS and a minor CS content on barium acetate gel. However, isolated GAGs appeared resistant to enzymatic digestion by heparinases. We found that these GAG extracts were heavily contaminated with RNA, which co-migrated with HS in barium acetate gel electrophoresis and interfered with 1,9-dimethylmethylene blue (DMMB) assays, resulting in an overestimation of GAG yields. We hypothesized that RNA may be contaminating GAG extracts from other cell cultures and possibly tissue, and therefore investigated potential RNA contaminations in GAG extracts from two additional cell lines, human umbilical vein endothelial cells and retinal pigmental epithelial cells, and mouse kidney, liver, spleen and heart tissue. GAG extracts from all examined cell lines and tissues contained varying amounts of contaminating RNA, which interfered with GAG quantification using DMMB assays and characterization of GAGs by barium acetate gel electrophoresis. We therefore recommend routinely evaluating the RNA content of GAG extracts and propose a robust protocol for GAG isolation that includes an RNA digestion step. PMID:27898729

  10. The Effect of Nucleic Acid Extraction Platforms and Sample Storage on the Integrity of Viral RNA for Use in Whole Genome Sequencing.

    PubMed

    Lewandowski, Kuiama; Bell, Andrew; Miles, Rory; Carne, Simon; Wooldridge, David; Manso, Carmen; Hennessy, Nicola; Bailey, Daniel; Pullan, Steven T; Gharbia, Saheer; Vipond, Richard

    2017-03-01

    Extraction of viral RNA and the storage of sample material are extremely important factors in the detection and whole genome sequencing (WGS) of viral pathogens. Although PCR-based detection methods focus on small amplicons, viral WGS applications require RNA of high quality and integrity for adequate sequence coverage and depth. This study examined the fitness of one manual and four automated RNA extraction platforms commonly used in diagnostic laboratories for use in metagenomic sequencing, how the practice of storing sample material in Qiagen buffer AVL before extraction affected the integrity of viral RNA and its suitability for use in amplicon-based WGS methods, and how the addition of Triton X-100 to buffer AVL affected the capability of the extraction platforms and the integrity of viral RNA in stored samples. This study found that the EZ1 platform gave the best performance of the automated platforms and gave comparable results to the frequently used manual Qiagen extraction protocol when extracted viral RNA was used in metagenomics sequencing. To maintain high levels of viral RNA integrity suitable for amplicon-based WGS, nucleic acid should be extracted from samples immediately, because even short storage periods in buffer AVL have a severe effect on integrity, and the addition of Triton X-100 had little effect on the quality of viral material for WGS. Crown Copyright © 2017. Published by Elsevier Inc. All rights reserved.

  11. Assessing an improved protocol for plasma microRNA extraction.

    PubMed

    Moret, Inés; Sánchez-Izquierdo, Dolors; Iborra, Marisa; Tortosa, Luis; Navarro-Puche, Ana; Nos, Pilar; Cervera, José; Beltrán, Belén

    2013-01-01

    The first step in biomarkers discovery is to identify the best protocols for their purification and analysis. This issue is critical when considering peripheral blood samples (plasma and serum) that are clinically interesting but meet several methodological problems, mainly complexity and low biomarker concentration. Analysis of small molecules, such as circulating microRNAs, should overcome these disadvantages. The present study describes an optimal RNA extraction method of microRNAs from human plasma samples. Different reagents and commercially available kits have been analyzed, identifying also the best pre-analytical conditions for plasma isolation. Between all of them, the column-based approaches were shown to be the most effective. In this context, miRNeasy Serum/Plasma Kit (from Qiagen) rendered more concentrated RNA, that was better suited for microarrays studies and did not require extra purification steps for sample concentration and purification than phenol based extraction methods. We also present evidences that the addition of low doses of an RNA carrier before starting the extraction process improves microRNA purification while an already published carrier dose can result in significant bias over microRNA profiles. Quality controls for best protocol selection were developed by spectrophotometry measurement of contaminants and microfluidics electrophoresis (Agilent 2100 Bioanalyzer) for RNA integrity. Selected donor and patient plasma samples and matched biopsies were tested by Affymetrix microarray technology to compare differentially expressed microRNAs. In summary, this study defines an optimized protocol for microRNA purification from human blood samples, increasing the performance of assays and shedding light over the best way to discover and use these biomarkers in clinical practice.

  12. SPARTA: Simple Program for Automated reference-based bacterial RNA-seq Transcriptome Analysis.

    PubMed

    Johnson, Benjamin K; Scholz, Matthew B; Teal, Tracy K; Abramovitch, Robert B

    2016-02-04

    Many tools exist in the analysis of bacterial RNA sequencing (RNA-seq) transcriptional profiling experiments to identify differentially expressed genes between experimental conditions. Generally, the workflow includes quality control of reads, mapping to a reference, counting transcript abundance, and statistical tests for differentially expressed genes. In spite of the numerous tools developed for each component of an RNA-seq analysis workflow, easy-to-use bacterially oriented workflow applications to combine multiple tools and automate the process are lacking. With many tools to choose from for each step, the task of identifying a specific tool, adapting the input/output options to the specific use-case, and integrating the tools into a coherent analysis pipeline is not a trivial endeavor, particularly for microbiologists with limited bioinformatics experience. To make bacterial RNA-seq data analysis more accessible, we developed a Simple Program for Automated reference-based bacterial RNA-seq Transcriptome Analysis (SPARTA). SPARTA is a reference-based bacterial RNA-seq analysis workflow application for single-end Illumina reads. SPARTA is turnkey software that simplifies the process of analyzing RNA-seq data sets, making bacterial RNA-seq analysis a routine process that can be undertaken on a personal computer or in the classroom. The easy-to-install, complete workflow processes whole transcriptome shotgun sequencing data files by trimming reads and removing adapters, mapping reads to a reference, counting gene features, calculating differential gene expression, and, importantly, checking for potential batch effects within the data set. SPARTA outputs quality analysis reports, gene feature counts and differential gene expression tables and scatterplots. SPARTA provides an easy-to-use bacterial RNA-seq transcriptional profiling workflow to identify differentially expressed genes between experimental conditions. This software will enable microbiologists with

  13. Extraction of MS2 Phage RNA from Upper Respiratory Tract Specimens by Use of Flat Glass Devices▿†

    PubMed Central

    Nanassy, Oliver Z.; Haydock, Paul; Beck, Nicola; Barker, Lynn M.; Hargrave, Perry; Gestwick, Daniel; Lindsey, Wesley C.; Reed, Michael W.; Meschke, J. Scott

    2011-01-01

    The isolation of pure nucleic acids from clinical samples is a crucial step in the molecular diagnosis of viral infections by nucleic acid testing (NAT). In this study, novel flat glass devices (cards) were demonstrated to support the rapid and efficient extraction of nucleic acids from upper respiratory tract specimens (nasal washes and swabs). The performance of the nucleic acid extraction cards was directly compared to an existing standardized and automated platform for viral extraction from these types of specimens. The flowthrough card method improved the speed of nucleic acid purification and accommodated larger sample volumes in extraction of bacteriophage MS2 RNA from the various specimen matrices. The dynamic range and estimated sensitivity of the card extraction method for reverse transcriptase quantitative real-time PCR (RT-qPCR)-based detection approximate those of the standardized magnetic glass bead extraction method used in this study. PMID:21191051

  14. Comparison of manual and automated nucleic acid extraction from whole-blood samples.

    PubMed

    Riemann, Kathrin; Adamzik, Michael; Frauenrath, Stefan; Egensperger, Rupert; Schmid, Kurt W; Brockmeyer, Norbert H; Siffert, Winfried

    2007-01-01

    Nucleic acid extraction and purification from whole blood is a routine application in many laboratories. Automation of this procedure promises standardized sample treatment, a low error rate, and avoidance of contamination. The performance of the BioRobot M48 (Qiagen) and the manual QIAmp DNA Blood Mini Kit (Qiagen) was compared for the extraction of DNA from whole blood. The concentration and purity of the extracted DNAs were determined by spectrophotometry. Analytical sensitivity was assessed by common PCR and genotyping techniques. The quantity and quality of the generated DNAs were slightly higher using the manual extraction method. The results of downstream applications were comparable to each other. Amplification of high-molecular-weight PCR fragments, genotyping by restriction digest, and pyrosequencing were successful for all samples. No cross-contamination could be detected. While automated DNA extraction requires significantly less hands-on time, it is slightly more expensive than the manual extraction method.

  15. Disposable and removable nucleic acid extraction and purification cartridges for automated flow-through systems

    DOEpatents

    Regan, John Frederick

    2014-09-09

    Removable cartridges are used on automated flow-through systems for the purpose of extracting and purifying genetic material from complex matrices. Different types of cartridges are paired with specific automated protocols to concentrate, extract, and purifying pathogenic or human genetic material. Their flow-through nature allows large quantities sample to be processed. Matrices may be filtered using size exclusion and/or affinity filters to concentrate the pathogen of interest. Lysed material is ultimately passed through a filter to remove the insoluble material before the soluble genetic material is delivered past a silica-like membrane that binds the genetic material, where it is washed, dried, and eluted. Cartridges are inserted into the housing areas of flow-through automated instruments, which are equipped with sensors to ensure proper placement and usage of the cartridges. Properly inserted cartridges create fluid- and air-tight seals with the flow lines of an automated instrument.

  16. An Automated Microwell Platform for Large-Scale Single Cell RNA-Seq

    PubMed Central

    Yuan, Jinzhou; Sims, Peter A.

    2016-01-01

    Recent developments have enabled rapid, inexpensive RNA sequencing of thousands of individual cells from a single specimen, raising the possibility of unbiased and comprehensive expression profiling from complex tissues. Microwell arrays are a particularly attractive microfluidic platform for single cell analysis due to their scalability, cell capture efficiency, and compatibility with imaging. We report an automated microwell array platform for single cell RNA-Seq with significantly improved performance over previous implementations. We demonstrate cell capture efficiencies of >50%, compatibility with commercially available barcoded mRNA capture beads, and parallel expression profiling from thousands of individual cells. We evaluate the level of cross-contamination in our platform by both tracking fluorescent cell lysate in sealed microwells and with a human-mouse mixed species RNA-Seq experiment. Finally, we apply our system to comprehensively assess heterogeneity in gene expression of patient-derived glioma neurospheres and uncover subpopulations similar to those observed in human glioma tissue. PMID:27670648

  17. An Automated Microwell Platform for Large-Scale Single Cell RNA-Seq.

    PubMed

    Yuan, Jinzhou; Sims, Peter A

    2016-09-27

    Recent developments have enabled rapid, inexpensive RNA sequencing of thousands of individual cells from a single specimen, raising the possibility of unbiased and comprehensive expression profiling from complex tissues. Microwell arrays are a particularly attractive microfluidic platform for single cell analysis due to their scalability, cell capture efficiency, and compatibility with imaging. We report an automated microwell array platform for single cell RNA-Seq with significantly improved performance over previous implementations. We demonstrate cell capture efficiencies of >50%, compatibility with commercially available barcoded mRNA capture beads, and parallel expression profiling from thousands of individual cells. We evaluate the level of cross-contamination in our platform by both tracking fluorescent cell lysate in sealed microwells and with a human-mouse mixed species RNA-Seq experiment. Finally, we apply our system to comprehensively assess heterogeneity in gene expression of patient-derived glioma neurospheres and uncover subpopulations similar to those observed in human glioma tissue.

  18. RNA 3'-terminal phosphate cyclase activity and RNA ligation in HeLa cell extract.

    PubMed Central

    Filipowicz, W; Konarska, M; Gross, H J; Shatkin, A J

    1983-01-01

    HeLa cell extract contains RNA ligase activity that converts linear polyribonucleotides to covalently closed circles. RNA substrates containing 2',3'-cyclic phosphate and 5'-hydroxyl termini are circularized by formation of a normal 3',5' phosphodiester bond. This activity differs from a previously described wheat germ RNA ligase which circularizes molecules with 2',3'-cyclic and 5' phosphate ends by a 2'-phosphomonester, 3',5'-phosphodiester linkage (Konarska et al., Nature 293, 112-116, 1981; Proc. Natl. Acad. Sci. USA 79, 1474-1478, 1982). The HeLa cell ligase can also utilize molecules with 3'-phosphate ends. However, in this case ligation is preceded by an ATP-dependent conversion of the 3'-terminal phosphate to the 2',3' cyclic form by a novel activity, RNA 3'-terminal phosphate cyclase. Both RNA ligase and RNA 3'-terminal phosphate cyclase activities are also present in extract of Xenopus oocyte nuclei, consistent with a role in RNA processing. Images PMID:6828385

  19. An automated workflow for quantifying RNA transcripts in individual cells in large data-sets.

    PubMed

    Pharris, Matthew C; Wu, Tzu-Ching; Chen, Xinping; Wang, Xu; Umulis, David M; Weake, Vikki M; Kinzer-Ursem, Tamara L

    2017-01-01

    Advanced molecular probing techniques such as single molecule fluorescence in situ hybridization (smFISH) or RNAscope can be used to assess the quantity and spatial location of mRNA transcripts within cells. Quantifying mRNA expression in large image sets usually involves automated counting of fluorescent spots. Though conventional spot counting algorithms may suffice, they often lack high-throughput capacity and accuracy in cases of crowded signal or excessive noise. Automatic identification of cells and processing of many images is still a challenge. We have developed a method to perform automatic cell boundary identification while providing quantitative data about mRNA transcript levels across many images. Comparisons of mRNA transcript levels identified by the method highly correlate to qPCR measurements of mRNA expression in Drosophila genotypes with different levels of Rhodopsin 1 transcript. We also introduce a graphical user interface to facilitate analysis of large data sets. We expect these methods to translate to model systems where automated image processing can be harnessed to obtain single-cell data. The described method: •Provides relative intensity measurements that scale directly with the number of labeled transcript probes within individual cells.•Allows quantitative assessment of single molecule data from images with crowded signal and moderate signal to noise ratios.

  20. Evaluation of four automated protocols for extraction of DNA from FTA cards.

    PubMed

    Stangegaard, Michael; Børsting, Claus; Ferrero-Miliani, Laura; Frank-Hansen, Rune; Poulsen, Lena; Hansen, Anders J; Morling, Niels

    2013-10-01

    Extraction of DNA using magnetic bead-based techniques on automated DNA extraction instruments provides a fast, reliable, and reproducible method for DNA extraction from various matrices. Here, we have compared the yield and quality of DNA extracted from FTA cards using four automated extraction protocols on three different instruments. The extraction processes were repeated up to six times with the same pieces of FTA cards. The sample material on the FTA cards was either blood or buccal cells. With the QIAamp DNA Investigator and QIAsymphony DNA Investigator kits, it was possible to extract DNA from the FTA cards in all six rounds of extractions in sufficient amount and quality to obtain complete short tandem repeat (STR) profiles on a QIAcube and a QIAsymphony SP. With the PrepFiler Express kit, almost all the extractable DNA was extracted in the first two rounds of extractions. Furthermore, we demonstrated that it was possible to successfully extract sufficient DNA for STR profiling from previously processed FTA card pieces that had been stored at 4 °C for up to 1 year. This showed that rare or precious FTA card samples may be saved for future analyses even though some DNA was already extracted from the FTA cards.

  1. Molecular Techniques for Dicistrovirus Detection without RNA Extraction or Purification

    PubMed Central

    Querido, Jailson F. B.; Agirre, Jon; Marti, Gerardo A.; Guérin, Diego M. A.; Silva, Marcelo Sousa

    2013-01-01

    Dicistroviridae is a new family of small, nonenveloped, and +ssRNA viruses pathogenic to both beneficial arthropods and insect pests as well. Triatoma virus (TrV), a dicistrovirus, is a pathogen of Triatoma infestans (Hemiptera: Reduviidae), one of the main vectors of Chagas disease. In this work, we report a single-step method to identify TrV, a dicistrovirus, isolated from fecal samples of triatomines. The identification method proved to be quite sensitive, even without the extraction and purification of RNA virus. PMID:23710438

  2. CompaRNA: a server for continuous benchmarking of automated methods for RNA secondary structure prediction

    PubMed Central

    Puton, Tomasz; Kozlowski, Lukasz P.; Rother, Kristian M.; Bujnicki, Janusz M.

    2013-01-01

    We present a continuous benchmarking approach for the assessment of RNA secondary structure prediction methods implemented in the CompaRNA web server. As of 3 October 2012, the performance of 28 single-sequence and 13 comparative methods has been evaluated on RNA sequences/structures released weekly by the Protein Data Bank. We also provide a static benchmark generated on RNA 2D structures derived from the RNAstrand database. Benchmarks on both data sets offer insight into the relative performance of RNA secondary structure prediction methods on RNAs of different size and with respect to different types of structure. According to our tests, on the average, the most accurate predictions obtained by a comparative approach are generated by CentroidAlifold, MXScarna, RNAalifold and TurboFold. On the average, the most accurate predictions obtained by single-sequence analyses are generated by CentroidFold, ContextFold and IPknot. The best comparative methods typically outperform the best single-sequence methods if an alignment of homologous RNA sequences is available. This article presents the results of our benchmarks as of 3 October 2012, whereas the rankings presented online are continuously updated. We will gladly include new prediction methods and new measures of accuracy in the new editions of CompaRNA benchmarks. PMID:23435231

  3. Spatial resolution requirements for automated cartographic road extraction

    USGS Publications Warehouse

    Benjamin, S.; Gaydos, L.

    1990-01-01

    Ground resolution requirements for detection and extraction of road locations in a digitized large-scale photographic database were investigated. A color infrared photograph of Sunnyvale, California was scanned, registered to a map grid, and spatially degraded to 1- to 5-metre resolution pixels. Road locations in each data set were extracted using a combination of image processing and CAD programs. These locations were compared to a photointerpretation of road locations to determine a preferred pixel size for the extraction method. Based on road pixel omission error computations, a 3-metre pixel resolution appears to be the best choice for this extraction method. -Authors

  4. RNA extraction from decaying wood for (meta)transcriptomic analyses.

    PubMed

    Adamo, Martino; Voyron, Samuele; Girlanda, Mariangela; Marmeisse, Roland

    2017-10-01

    Wood decomposition is a key step of the terrestrial carbon cycle and is of economic importance. It is essentially a microbiological process performed by fungi and to an unknown extent by bacteria. To gain access to the genes expressed by the diverse microbial communities participating in wood decay, we developed an RNA extraction protocol from this recalcitrant material rich in polysaccharides and phenolic compounds. This protocol was implemented on 22 wood samples representing as many tree species from 11 plant families in the Angiosperms and Gymnosperms. RNA was successfully extracted from all samples and converted into cDNAs from which were amplified both fungal and bacterial protein coding genes, including genes encoding hydrolytic enzymes participating in lignocellulose hydrolysis. This protocol applicable to a wide range of decomposing wood types represents a first step towards a metatranscriptomic analysis of wood degradation under natural conditions.

  5. RNA extraction from equine samples for equine influenza virus.

    PubMed

    Balasuriya, Udeni B R

    2014-01-01

    The primary goals of this chapter are to discuss common viral RNA isolation and purification methods that are routinely used by various diagnostic laboratories, to highlight the advantages and drawbacks of each method, and to identify the most suitable and reliable method to increase the sensitivity and specificity of RT-PCR assays for the detection of equine influenza virus (EIV) in clinical specimens. Our experiences and review of literature show that magnetic bead-based nucleic extraction methods (manual and automatic) work well for isolation and purification of EIV RNA from nasal swab specimens. Furthermore, most of the information presented in this chapter could be directly applicable to isolation and purification of nucleic acids (both DNA and RNA) from other equine clinical samples.

  6. RNA structure framework: automated transcriptome-wide reconstruction of RNA secondary structures from high-throughput structure probing data.

    PubMed

    Incarnato, Danny; Neri, Francesco; Anselmi, Francesca; Oliviero, Salvatore

    2016-02-01

    The rapidly increasing number of discovered non-coding RNAs makes the understanding of their structure a key feature toward a deeper comprehension of gene expression regulation. Various enzymatic- and chemically- based approaches have been recently developed to allow whole-genome studies of RNA secondary structures. Several methods have been recently presented that allow high-throughput RNA structure probing (CIRS-seq, Structure-seq, SHAPE-seq, PARS, etc.) and unbiased structural inference of residues within RNAs in their native conformation. We here present an analysis toolkit, named RNA Structure Framework (RSF), which allows fast and fully-automated analysis of high-throughput structure probing data, from data pre-processing to whole-transcriptome RNA structure inference. RSF is written in Perl and is freely available under the GPLv3 license from http://rsf.hugef-research.org. salvatore.oliviero@hugef-torino.org Supplementary data are available at Bioinformatics online. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  7. Feature Extraction and Selection Strategies for Automated Target Recognition

    NASA Technical Reports Server (NTRS)

    Greene, W. Nicholas; Zhang, Yuhan; Lu, Thomas T.; Chao, Tien-Hsin

    2010-01-01

    Several feature extraction and selection methods for an existing automatic target recognition (ATR) system using JPLs Grayscale Optical Correlator (GOC) and Optimal Trade-Off Maximum Average Correlation Height (OT-MACH) filter were tested using MATLAB. The ATR system is composed of three stages: a cursory region of-interest (ROI) search using the GOC and OT-MACH filter, a feature extraction and selection stage, and a final classification stage. Feature extraction and selection concerns transforming potential target data into more useful forms as well as selecting important subsets of that data which may aide in detection and classification. The strategies tested were built around two popular extraction methods: Principal Component Analysis (PCA) and Independent Component Analysis (ICA). Performance was measured based on the classification accuracy and free-response receiver operating characteristic (FROC) output of a support vector machine(SVM) and a neural net (NN) classifier.

  8. Feature Extraction and Selection Strategies for Automated Target Recognition

    NASA Technical Reports Server (NTRS)

    Greene, W. Nicholas; Zhang, Yuhan; Lu, Thomas T.; Chao, Tien-Hsin

    2010-01-01

    Several feature extraction and selection methods for an existing automatic target recognition (ATR) system using JPLs Grayscale Optical Correlator (GOC) and Optimal Trade-Off Maximum Average Correlation Height (OT-MACH) filter were tested using MATLAB. The ATR system is composed of three stages: a cursory region of-interest (ROI) search using the GOC and OT-MACH filter, a feature extraction and selection stage, and a final classification stage. Feature extraction and selection concerns transforming potential target data into more useful forms as well as selecting important subsets of that data which may aide in detection and classification. The strategies tested were built around two popular extraction methods: Principal Component Analysis (PCA) and Independent Component Analysis (ICA). Performance was measured based on the classification accuracy and free-response receiver operating characteristic (FROC) output of a support vector machine(SVM) and a neural net (NN) classifier.

  9. Automated insertion of sequences into a ribosomal RNA alignment: An application of computational linguistics in molecular biology

    SciTech Connect

    Taylor, Ronald C.

    1991-11-01

    This thesis involved the construction of (1) a grammar that incorporates knowledge on base invariancy and secondary structure in a molecule and (2) a parser engine that uses the grammar to position bases into the structural subunits of the molecule. These concepts were combined with a novel pinning technique to form a tool that semi-automates insertion of a new species into the alignment for the 16S rRNA molecule (a component of the ribosome) maintained by Dr. Carl Woese`s group at the University of Illinois at Urbana. The tool was tested on species extracted from the alignment and on a group of entirely new species. The results were very encouraging, and the tool should be substantial aid to the curators of the 16S alignment. The construction of the grammar was itself automated, allowing application of the tool to alignments for other molecules. The logic programming language Prolog was used to construct all programs involved. The computational linguistics approach used here was found to be a useful way to attach the problem of insertion into an alignment.

  10. Automated insertion of sequences into a ribosomal RNA alignment: An application of computational linguistics in molecular biology

    SciTech Connect

    Taylor, R.C.

    1991-11-01

    This thesis involved the construction of (1) a grammar that incorporates knowledge on base invariancy and secondary structure in a molecule and (2) a parser engine that uses the grammar to position bases into the structural subunits of the molecule. These concepts were combined with a novel pinning technique to form a tool that semi-automates insertion of a new species into the alignment for the 16S rRNA molecule (a component of the ribosome) maintained by Dr. Carl Woese's group at the University of Illinois at Urbana. The tool was tested on species extracted from the alignment and on a group of entirely new species. The results were very encouraging, and the tool should be substantial aid to the curators of the 16S alignment. The construction of the grammar was itself automated, allowing application of the tool to alignments for other molecules. The logic programming language Prolog was used to construct all programs involved. The computational linguistics approach used here was found to be a useful way to attach the problem of insertion into an alignment.

  11. Applicability of a System for fully automated nucleic acid extraction from formalin-fixed paraffin-embedded sections for routine KRAS mutation testing.

    PubMed

    Lehmann, Annika; Schewe, Christiane; Hennig, Guido; Denkert, Carsten; Weichert, Wilko; Budczies, Jan; Dietel, Manfred

    2012-06-01

    Due to the approval of various new targeted therapies for the treatment of cancer, molecular pathology laboratories with a diagnostic focus have to meet new challenges: simultaneous handling of a large number of samples, small amounts of input material, and fragmentation of nucleic acids because of formalin fixation. As a consequence, fully automated systems for a fast and standardized extraction of high-quality DNA from formalin-fixed paraffin-embedded (FFPE) tissues are urgently needed. In this study, we tested the performance of a fully automated, high-throughput method for the extraction of nucleic acids from FFPE tissues. We investigated the extraction performance in sections of 5 different tissue types often analyzed in routine pathology laboratories (cervix, colon, liver, lymph node, and lung; n=340). Furthermore, we compared the quality, labor input, and applicability of the method for diagnostic purposes with those of a laboratory-validated manual method in a clinical setting by screening a set of 45 colorectal adenocarcinoma for the KRAS mutation. Automated extraction of both DNA and RNA was successful in 339 of 340 FFPE samples representing 5 different tissue types. In comparison with a conventional manual extraction protocol, the method showed an overall agreement of 97.7% (95% confidence interval, 88.2%-99.9%) for the subsequent mutational analysis of the KRAS gene in colorectal cancer samples. The fully automated system is a promising tool for a simple, robust, and rapid extraction of DNA and RNA from formalin-fixed tissue. It ensures a standardization of sample processing and can be applied to clinical FFPE samples in routine pathology.

  12. Automation and Other Extensions of the SMAC Modal Parameter Extraction Package

    SciTech Connect

    KLENKE,SCOTT E.; MAYES,RANDALL L.

    1999-11-01

    As model validation techniques gain more acceptance and increase in power, the demands on the modal parameter extractions increase. The estimation accuracy, the number of modes desired, and the data reduction efficiency are required features. An algorithm known as SMAC (Synthesize Modes And Correlate), based on principles of modal filtering, has been in development for a few years. SMAC has now been extended in two main areas. First, it has now been automated. Second, it has been extended to fit complex modes as well as real modes. These extensions have enhanced the power of modal extraction so that, typically, the analyst needs to manually fit only 10 percent of the modes in the desired bandwidth, whereas the automated routines will fit 90 percent of the modes. SMAC could be successfully automated because it generally does not produce computational roots.

  13. Automated Protein Biomarker Analysis: on-line extraction of clinical samples by Molecularly Imprinted Polymers

    PubMed Central

    Rossetti, Cecilia; Świtnicka-Plak, Magdalena A.; Grønhaug Halvorsen, Trine; Cormack, Peter A.G.; Sellergren, Börje; Reubsaet, Léon

    2017-01-01

    Robust biomarker quantification is essential for the accurate diagnosis of diseases and is of great value in cancer management. In this paper, an innovative diagnostic platform is presented which provides automated molecularly imprinted solid-phase extraction (MISPE) followed by liquid chromatography-mass spectrometry (LC-MS) for biomarker determination using ProGastrin Releasing Peptide (ProGRP), a highly sensitive biomarker for Small Cell Lung Cancer, as a model. Molecularly imprinted polymer microspheres were synthesized by precipitation polymerization and analytical optimization of the most promising material led to the development of an automated quantification method for ProGRP. The method enabled analysis of patient serum samples with elevated ProGRP levels. Particularly low sample volumes were permitted using the automated extraction within a method which was time-efficient, thereby demonstrating the potential of such a strategy in a clinical setting. PMID:28303910

  14. Automated Protein Biomarker Analysis: on-line extraction of clinical samples by Molecularly Imprinted Polymers

    NASA Astrophysics Data System (ADS)

    Rossetti, Cecilia; Świtnicka-Plak, Magdalena A.; Grønhaug Halvorsen, Trine; Cormack, Peter A. G.; Sellergren, Börje; Reubsaet, Léon

    2017-03-01

    Robust biomarker quantification is essential for the accurate diagnosis of diseases and is of great value in cancer management. In this paper, an innovative diagnostic platform is presented which provides automated molecularly imprinted solid-phase extraction (MISPE) followed by liquid chromatography-mass spectrometry (LC-MS) for biomarker determination using ProGastrin Releasing Peptide (ProGRP), a highly sensitive biomarker for Small Cell Lung Cancer, as a model. Molecularly imprinted polymer microspheres were synthesized by precipitation polymerization and analytical optimization of the most promising material led to the development of an automated quantification method for ProGRP. The method enabled analysis of patient serum samples with elevated ProGRP levels. Particularly low sample volumes were permitted using the automated extraction within a method which was time-efficient, thereby demonstrating the potential of such a strategy in a clinical setting.

  15. Labeling of multiple cell markers and mRNA using automated apparatus.

    PubMed

    Paterson, Jennifer C; Ballabio, Erica; Mattsson, Göran; Turner, Susan H; Mason, David Y; Marafioti, Teresa

    2008-07-01

    Double immunoenzymatic labeling of 2 different molecules in tissue sections is a widely used technique. However, it is time consuming since the 2 immunoenzymatic procedures are carried out in sequence, and they must also be optimally performed to avoid unwanted background labeling. In this paper, we report that double immunoenzymatic staining performed using automated immunostaining apparatus considerably reduces the requirements in terms of time and is also highly reproducible and free of background. Three tissue markers can also be visualized by performing (after immunoperoxidase labeling) 2 sequential immuno-alkaline phosphatase procedures using different substrates. Furthermore, single or double detection of mRNA by in situ hybridization can be combined with immunoenzymatic labeling. Finally, automated labeling could also be performed on peripheral blood and bone marrow smears, opening the possibility of using this procedure in the analysis of hematologic/cytology samples.

  16. Evaluation of an automated high-volume extraction method for viral nucleic acids in comparison to a manual procedure with preceding enrichment.

    PubMed

    Hourfar, M K; Schmidt, M; Seifried, E; Roth, W K

    2005-08-01

    Nucleic acid extraction still harbours the potential for improvements in automation and sensitivity of nucleic acid amplification technology (NAT) testing. This study evaluates the feasibility of a novel automated high-volume extraction protocol for NAT minipool testing in a blood bank setting. The chemagic Viral DNA/RNA Kit special for automated purification of viral nucleic acids from 9.6 ml of plasma by using the chemagic Magnetic Separation Module I was investigated. Analytical sensitivity for hepatitis C virus (HCV), human immunodeficiency virus-1 (HIV-1), hepatitis B virus (HBV), hepatitis A virus (HAV) and parvovirus B19 (B19) was compared to our present manual procedure that involves virus enrichment by centrifugation. Chemagic technology allows automation of the viral DNA/RNA extraction process. Viral nucleic acids were bound directly to magnetic beads from 9.6-ml minipools. By combining the automated magnetic beads-based extraction technology with our in-house TaqMan polymerase chain reaction (PCR) assays, 95% detection limits were 280 IU/ml for HCV, 4955 IU/ml for HIV-1, 249 IU/ml for HBV, 462 IU/ml for HAV and 460 IU/ml for B19, calculated for an individual donation in a pool of 96 donors. The detection limits of our present method were 460 IU/ml for HCV, 879 IU/ml for HIV-1, 90 IU/ml for HBV, 203 IU/ml for HAV and 314 IU/ml for B19. The 95% detection limits obtained by using the chemagic method were within the regulatory requirements for blood donor screening. The sensitivities detected for HCV, HBV, HAV and B19 were found to be in a range similar to that of the manual purification method. Sensitivity for HIV-1, however, was found to be inferior for the chemagic method in this study.

  17. Computational strategies for the automated design of RNA nanoscale structures from building blocks using NanoTiler☆

    PubMed Central

    Bindewald, Eckart; Grunewald, Calvin; Boyle, Brett; O’Connor, Mary; Shapiro, Bruce A.

    2013-01-01

    One approach to designing RNA nanoscale structures is to use known RNA structural motifs such as junctions, kissing loops or bulges and to construct a molecular model by connecting these building blocks with helical struts. We previously developed an algorithm for detecting internal loops, junctions and kissing loops in RNA structures. Here we present algorithms for automating or assisting many of the steps that are involved in creating RNA structures from building blocks: (1) assembling building blocks into nanostructures using either a combinatorial search or constraint satisfaction; (2) optimizing RNA 3D ring structures to improve ring closure; (3) sequence optimisation; (4) creating a unique non-degenerate RNA topology descriptor. This effectively creates a computational pipeline for generating molecular models of RNA nanostructures and more specifically RNA ring structures with optimized sequences from RNA building blocks. We show several examples of how the algorithms can be utilized to generate RNA tecto-shapes. PMID:18838281

  18. Discovering Indicators of Successful Collaboration Using Tense: Automated Extraction of Patterns in Discourse

    ERIC Educational Resources Information Center

    Thompson, Kate; Kennedy-Clark, Shannon; Wheeler, Penny; Kelly, Nick

    2014-01-01

    This paper describes a technique for locating indicators of success within the data collected from complex learning environments, proposing an application of e-research to access learner processes and measure and track group progress. The technique combines automated extraction of tense and modality via parts-of-speech tagging with a visualisation…

  19. Discovering Indicators of Successful Collaboration Using Tense: Automated Extraction of Patterns in Discourse

    ERIC Educational Resources Information Center

    Thompson, Kate; Kennedy-Clark, Shannon; Wheeler, Penny; Kelly, Nick

    2014-01-01

    This paper describes a technique for locating indicators of success within the data collected from complex learning environments, proposing an application of e-research to access learner processes and measure and track group progress. The technique combines automated extraction of tense and modality via parts-of-speech tagging with a visualisation…

  20. Ebbie: automated analysis and storage of small RNA cloning data using a dynamic web server.

    PubMed

    Ebhardt, H Alexander; Wiese, Kay C; Unrau, Peter J

    2006-04-03

    DNA sequencing is used ubiquitously: from deciphering genomes to determining the primary sequence of small RNAs (smRNAs). The cloning of smRNAs is currently the most conventional method to determine the actual sequence of these important regulators of gene expression. Typical smRNA cloning projects involve the sequencing of hundreds to thousands of smRNA clones that are delimited at their 5' and 3' ends by fixed sequence regions. These primers result from the biochemical protocol used to isolate and convert the smRNA into clonable PCR products. Recently we completed a smRNA cloning project involving tobacco plants, where analysis was required for approximately 700 smRNA sequences. Finding no easily accessible research tool to enter and analyze smRNA sequences we developed Ebbie to assist us with our study. Ebbie is a semi-automated smRNA cloning data processing algorithm, which initially searches for any substring within a DNA sequencing text file, which is flanked by two constant strings. The substring, also termed smRNA or insert, is stored in a MySQL and BlastN database. These inserts are then compared using BlastN to locally installed databases allowing the rapid comparison of the insert to both the growing smRNA database and to other static sequence databases. Our laboratory used Ebbie to analyze scores of DNA sequencing data originating from an smRNA cloning project. Through its built-in instant analysis of all inserts using BlastN, we were able to quickly identify 33 groups of smRNAs from approximately 700 database entries. This clustering allowed the easy identification of novel and highly expressed clusters of smRNAs. Ebbie is available under GNU GPL and currently implemented on http://bioinformatics.org/ebbie/. Ebbie was designed for medium sized smRNA cloning projects with about 1,000 database entries. Ebbie can be used for any type of sequence analysis where two constant primer regions flank a sequence of interest. The reliable storage of inserts, and

  1. Automated extraction of pleural effusion in three-dimensional thoracic CT images

    NASA Astrophysics Data System (ADS)

    Kido, Shoji; Tsunomori, Akinori

    2009-02-01

    It is important for diagnosis of pulmonary diseases to measure volume of accumulating pleural effusion in threedimensional thoracic CT images quantitatively. However, automated extraction of pulmonary effusion correctly is difficult. Conventional extraction algorithm using a gray-level based threshold can not extract pleural effusion from thoracic wall or mediastinum correctly, because density of pleural effusion in CT images is similar to those of thoracic wall or mediastinum. So, we have developed an automated extraction method of pulmonary effusion by use of extracting lung area with pleural effusion. Our method used a template of lung obtained from a normal lung for segmentation of lungs with pleural effusions. Registration process consisted of two steps. First step was a global matching processing between normal and abnormal lungs of organs such as bronchi, bones (ribs, sternum and vertebrae) and upper surfaces of livers which were extracted using a region-growing algorithm. Second step was a local matching processing between normal and abnormal lungs which were deformed by the parameter obtained from the global matching processing. Finally, we segmented a lung with pleural effusion by use of the template which was deformed by two parameters obtained from the global matching processing and the local matching processing. We compared our method with a conventional extraction method using a gray-level based threshold and two published methods. The extraction rates of pleural effusions obtained from our method were much higher than those obtained from other methods. Automated extraction method of pulmonary effusion by use of extracting lung area with pleural effusion is promising for diagnosis of pulmonary diseases by providing quantitative volume of accumulating pleural effusion.

  2. Comparison of commercial RNA extraction kits for preparation of DNA-free total RNA from Salmonella cells.

    PubMed

    Rump, Lydia V; Asamoah, Benedicta; Gonzalez-Escalona, Narjol

    2010-07-27

    The isolation of DNA-free RNA is a crucial step in the reverse transcription PCR (RT-PCR). Every RNA extraction procedure results in RNA samples contaminated with genomic DNA, which can cause false-positive outcomes in highly sensitive applications, including a recently developed quantitative real-time PCR (RT-qPCR) assay that targets invA mRNA for the detection of live Salmonella cells. The assay of this specific mRNA can be used to indicate the presence of live, as opposed to dead, cells of Salmonella enterica in a food matrix. We evaluated the ability of five RNA extraction kits to produce RNA preparations from exponentially growing Salmonella cells. The acceptability of the preparations for use in downstream applications such as RT-qPCR was judged in terms of the total amount of RNA recovered, the integrity of the RNA molecules, and minimal content of DNA. The five kits produced RNA preparations that differed markedly in yield, integrity of the Salmonella RNA and the amount of contaminant DNA. The greatest RNA recovery was achieved with the MasterPure kit; however, the preparation contained high levels of genomic DNA. The UltraClean extraction kit gave a low level of RNA recovery with a poor level of integrity. The RNeasy Mini, RiboPure and PureLink extraction kits produced high-quality, DNA-free RNA suitable for Salmonella detection by RT-qPCR. We showed that the RNeasy Mini and PureLink RNA extraction kits were the most suitable for the detection of Salmonella invA mRNA by RT-qPCR. The use of these two kits will greatly reduce the frequency of false-positive results and might allow fast RT-qPCR determination of invA mRNA produced by viable Salmonella in food samples.

  3. De novo automated design of small RNA circuits for engineering synthetic riboregulation in living cells

    PubMed Central

    Rodrigo, Guillermo; Landrain, Thomas E.; Jaramillo, Alfonso

    2012-01-01

    A grand challenge in synthetic biology is to use our current knowledge of RNA science to perform the automatic engineering of completely synthetic sequences encoding functional RNAs in living cells. We report here a fully automated design methodology and experimental validation of synthetic RNA interaction circuits working in a cellular environment. The computational algorithm, based on a physicochemical model, produces novel RNA sequences by exploring the space of possible sequences compatible with predefined structures. We tested our methodology in Escherichia coli by designing several positive riboregulators with diverse structures and interaction models, suggesting that only the energy of formation and the activation energy (free energy barrier to overcome for initiating the hybridization reaction) are sufficient criteria to engineer RNA interaction and regulation in bacteria. The designed sequences exhibit nonsignificant similarity to any known noncoding RNA sequence. Our riboregulatory devices work independently and in combination with transcription regulation to create complex logic circuits. Our results demonstrate that a computational methodology based on first-principles can be used to engineer interacting RNAs with allosteric behavior in living cells. PMID:22949707

  4. Plant RNA processing: soybean pre-mRNA in a pea cell-free extract

    SciTech Connect

    Schuler, M.A.; Hanley, B.A.

    1987-05-01

    Using a pea cell-free extract they have demonstrated the splicing of an SP6 fusion transcript containing an intron derived from the soybean seed storage protein ..beta..-subunit gene. Intron 115 from the conglycinin gene was cloned into a SP6 vector and transcribed using standard recombinant DNA techniques. Incubation of radioactively labeled fusion transcripts in the cell-free system produced a number of products which were identified by primer extension and S1 nuclease analysis. All the products are linear RNA molecules. Lariat intermediates, similar to those found in the yeast and HeLa cell RNA processing systems, have not been detected. The linear RNA products detected in their plant in vitro processing system have various portions of the intron removed which suggests that alternative splice sites are used in processing of this plant intron due to activation of cryptic splice sites or creation of splice sites in the fusion construction. The kinetics of the reactions and parameters of the extract are similar to those determined for the HeLa cell system. Sucrose gradient analysis has demonstrated that the plant RNA products sedimented in a 30S particle, similar in size to that found for the spliceosome of the HeLa cell system.

  5. Towards automated support for extraction of reusable components

    NASA Technical Reports Server (NTRS)

    Abd-El-hafiz, S. K.; Basili, Victor R.; Caldiera, Gianluigi

    1992-01-01

    A cost effective introduction of software reuse techniques requires the reuse of existing software developed in many cases without aiming at reusability. This paper discusses the problems related to the analysis and reengineering of existing software in order to reuse it. We introduce a process model for component extraction and focus on the problem of analyzing and qualifying software components which are candidates for reuse. A prototype tool for supporting the extraction of reusable components is presented. One of the components of this tool aids in understanding programs and is based on the functional model of correctness. It can assist software engineers in the process of finding correct formal specifications for programs. A detailed description of this component and an example to demonstrate a possible operational scenario are given.

  6. Data Mining: The Art of Automated Knowledge Extraction

    NASA Astrophysics Data System (ADS)

    Karimabadi, H.; Sipes, T.

    2012-12-01

    Data mining algorithms are used routinely in a wide variety of fields and they are gaining adoption in sciences. The realities of real world data analysis are that (a) data has flaws, and (b) the models and assumptions that we bring to the data are inevitably flawed, and/or biased and misspecified in some way. Data mining can improve data analysis by detecting anomalies in the data, check for consistency of the user model assumptions, and decipher complex patterns and relationships that would not be possible otherwise. The common form of data collected from in situ spacecraft measurements is multi-variate time series which represents one of the most challenging problems in data mining. We have successfully developed algorithms to deal with such data and have extended the algorithms to handle streaming data. In this talk, we illustrate the utility of our algorithms through several examples including automated detection of reconnection exhausts in the solar wind and flux ropes in the magnetotail. We also show examples from successful applications of our technique to analysis of 3D kinetic simulations. With an eye to the future, we provide an overview of our upcoming plans that include collaborative data mining, expert outsourcing data mining, computer vision for image analysis, among others. Finally, we discuss the integration of data mining algorithms with web-based services such as VxOs and other Heliophysics data centers and the resulting capabilities that it would enable.

  7. Automated extraction and variability analysis of sulcal neuroanatomy.

    PubMed

    Le Goualher, G; Procyk, E; Collins, D L; Venugopal, R; Barillot, C; Evans, A C

    1999-03-01

    Systematic mapping of the variability in cortical sulcal anatomy is an area of increasing interest which presents numerous methodological challenges. To address these issues, we have implemented sulcal extraction and assisted labeling (SEAL) to automatically extract the two-dimensional (2-D) surface ribbons that represent the median axis of cerebral sulci and to neuroanatomically label these entities. To encode the extracted three-dimensional (3-D) cortical sulcal schematic topography (CSST) we define a relational graph structure composed of two main features: vertices (representing sulci) and arcs (representing the relationships between sulci). Vertices contain a parametric representation of the surface ribbon buried within the sulcus. Points on this surface are expressed in stereotaxic coordinates (i.e., with respect to a standardized brain coordinate system). For each of these vertices, we store length, depth, and orientation as well as anatomical attributes (e.g., hemisphere, lobe, sulcus type, etc.). Each arc stores the 3-D location of the junction between sulci as well as a list of its connecting sulci. Sulcal labeling is performed semiautomatically by selecting a sulcal entity in the CSST and selecting from a menu of candidate sulcus names. In order to help the user in the labeling task, the menu is restricted to the most likely candidates by using priors for the expected sulcal spatial distribution. These priors, i.e., sulcal probabilistic maps, were created from the spatial distribution of 34 sulci traced manually on 36 different subjects. Given these spatial probability maps, the user is provided with the likelihood that the selected entity belongs to a particular sulcus. The cortical structure representation obtained by SEAL is suitable to extract statistical information about both the spatial and the structural composition of the cerebral cortical topography. This methodology allows for the iterative construction of a successively more complete

  8. Multispectral Image Road Extraction Based Upon Automated Map Conflation

    NASA Astrophysics Data System (ADS)

    Chen, Bin

    Road network extraction from remotely sensed imagery enables many important and diverse applications such as vehicle tracking, drone navigation, and intelligent transportation studies. There are, however, a number of challenges to road detection from an image. Road pavement material, width, direction, and topology vary across a scene. Complete or partial occlusions caused by nearby buildings, trees, and the shadows cast by them, make maintaining road connectivity difficult. The problems posed by occlusions are exacerbated with the increasing use of oblique imagery from aerial and satellite platforms. Further, common objects such as rooftops and parking lots are made of materials similar or identical to road pavements. This problem of common materials is a classic case of a single land cover material existing for different land use scenarios. This work addresses these problems in road extraction from geo-referenced imagery by leveraging the OpenStreetMap digital road map to guide image-based road extraction. The crowd-sourced cartography has the advantages of worldwide coverage that is constantly updated. The derived road vectors follow only roads and so can serve to guide image-based road extraction with minimal confusion from occlusions and changes in road material. On the other hand, the vector road map has no information on road widths and misalignments between the vector map and the geo-referenced image are small but nonsystematic. Properly correcting misalignment between two geospatial datasets, also known as map conflation, is an essential step. A generic framework requiring minimal human intervention is described for multispectral image road extraction and automatic road map conflation. The approach relies on the road feature generation of a binary mask and a corresponding curvilinear image. A method for generating the binary road mask from the image by applying a spectral measure is presented. The spectral measure, called anisotropy-tunable distance (ATD

  9. Automation of solid-phase microextraction-gas chromatography-mass spectrometry extraction of eucalyptus volatiles.

    PubMed

    Zini, Cláudia A; Lord, Heather; Christensen, Eva; de, Assis Teotĵnio F; Caramão, Elina B; Pawliszyn, Janusz

    2002-03-01

    Solid-phase microextraction (SPME) coupled with gas chromatography (GC)-ion-trap mass spectrometry (ITMS) is employed to analyze fragrance compounds from different species of eucalyptus trees: Eucalyptus dunnii, Eucalyptus saligna, Eucalyptus grandis, and hybrids of other species. The analyses are performed using an automated system for preincubation, extraction, injection, and analysis of samples. The autosampler used is a CombiPAL and has much flexibility for the development of SPME methods and accommodates a variety of vial sizes. For automated fragrance analysis the 10- and 20-mL vials are the most appropriate. The chromatographic separation and identification of the analytes are performed with a Varian Saturn 4D GC-ITMS using an HP-5MS capillary column. Several compounds of eucalyptus volatiles are identified, with good reproducibility for both the peak areas and retention times. Equilibrium extraction provides maximal sensitivity but requires additional consideration for the effect of carryover. Preequilibrium extraction allows good sensitivity with minimal carryover.

  10. Optimization of RNA Purification and Analysis for Automated, Pre-Symptomatic Disease Diagnostics

    SciTech Connect

    Vaidya, A; Nasarabadi, S; Milanovich, F

    2005-06-28

    When diagnosing disease, time is often a more formidable enemy than the pathogen itself. Current detection methods rely primarily on post-symptomatic protein production (i.e. antibodies), which does not occur in noticeable levels until several weeks after infection. As such, a major goal among researchers today is to expedite pre-symptomatic disease recognition and treatment. Since most pathogens are known to leave a unique signature on the genetic expression of the host, one potential diagnostic tool is host mRNA. In my experiments, I examined several methods of isolating RNA and reading its genetic sequence. I first used two types of reverse transcriptase polymerase chain reactions (using commercial RNA) and examined the resultant complementary DNA through gel electrophoresis. I then proceeded to isolate and purify whole RNA from actual human monocytes and THP-1 cells using several published methods, and examined gene expression on the RNA itself. I compared the two RT-PCR methods and concluded that a double step RT-PCR is superior to the single step method. I also compared the various techniques of RNA isolation by examining the yield and purity of the resultant RNA. Finally, I studied the level of cellular IL-8 and IL-1 gene expression, two genes involved in the human immune response, which can serve as a baseline for future genetic comparison with LPS-exposed cells. Based on the results, I have determined which conditions and procedures are optimal for RNA isolation, RT-PCR, and RNA yield assessment. The overall goal of my research is to develop a flow-through system of RNA analysis, whereby blood samples can be collected and analyzed for disease prior to the onset of symptoms. The Pathomics group hopes to automate this process by removing the human labor factor, thereby decreasing the procedure's cost and increasing its availability to the general population. Eventually, our aim is to have an autonomous diagnostic system based on RNA analysis that would

  11. Highly efficient automated extraction of DNA from old and contemporary skeletal remains.

    PubMed

    Zupanič Pajnič, Irena; Debska, Magdalena; Gornjak Pogorelc, Barbara; Vodopivec Mohorčič, Katja; Balažic, Jože; Zupanc, Tomaž; Štefanič, Borut; Geršak, Ksenija

    2016-01-01

    We optimised the automated extraction of DNA from old and contemporary skeletal remains using the AutoMate Express system and the PrepFiler BTA kit. 24 Contemporary and 25 old skeletal remains from WWII were analysed. For each skeleton, extraction using only 0.05 g of powder was performed according to the manufacturer's recommendations (no demineralisation - ND method). Since only 32% of full profiles were obtained from aged and 58% from contemporary casework skeletons, the extraction protocol was modified to acquire higher quality DNA and genomic DNA was obtained after full demineralisation (FD method). The nuclear DNA of the samples was quantified using the Investigator Quantiplex kit and STR typing was performed using the NGM kit to evaluate the performance of tested extraction methods. In the aged DNA samples, 64% of full profiles were obtained using the FD method. For the contemporary skeletal remains the performance of the ND method was closer to the FD method compared to the old skeletons, giving 58% of full profiles with the ND method and 71% of full profiles using the FD method. The extraction of DNA from only 0.05 g of bone or tooth powder using the AutoMate Express has proven highly successful in the recovery of DNA from old and contemporary skeletons, especially with the modified FD method. We believe that the results obtained will contribute to the possibilities of using automated devices for extracting DNA from skeletal remains, which would shorten the procedures for obtaining high-quality DNA from skeletons in forensic laboratories.

  12. Evaluation of three automated nucleic acid extraction systems for identification of respiratory viruses in clinical specimens by multiplex real-time PCR.

    PubMed

    Kim, Yoonjung; Han, Mi-Soon; Kim, Juwon; Kwon, Aerin; Lee, Kyung-A

    2014-01-01

    A total of 84 nasopharyngeal swab specimens were collected from 84 patients. Viral nucleic acid was extracted by three automated extraction systems: QIAcube (Qiagen, Germany), EZ1 Advanced XL (Qiagen), and MICROLAB Nimbus IVD (Hamilton, USA). Fourteen RNA viruses and two DNA viruses were detected using the Anyplex II RV16 Detection kit (Seegene, Republic of Korea). The EZ1 Advanced XL system demonstrated the best analytical sensitivity for all the three viral strains. The nucleic acids extracted by EZ1 Advanced XL showed higher positive rates for virus detection than the others. Meanwhile, the MICROLAB Nimbus IVD system was comprised of fully automated steps from nucleic extraction to PCR setup function that could reduce human errors. For the nucleic acids recovered from nasopharyngeal swab specimens, the QIAcube system showed the fewest false negative results and the best concordance rate, and it may be more suitable for detecting various viruses including RNA and DNA virus strains. Each system showed different sensitivity and specificity for detection of certain viral pathogens and demonstrated different characteristics such as turnaround time and sample capacity. Therefore, these factors should be considered when new nucleic acid extraction systems are introduced to the laboratory.

  13. Fully Automated Electro Membrane Extraction Autosampler for LC-MS Systems Allowing Soft Extractions for High-Throughput Applications.

    PubMed

    Fuchs, David; Pedersen-Bjergaard, Stig; Jensen, Henrik; Rand, Kasper D; Honoré Hansen, Steen; Petersen, Nickolaj Jacob

    2016-07-05

    The current work describes the implementation of electro membrane extraction (EME) into an autosampler for high-throughput analysis of samples by EME-LC-MS. The extraction probe was built into a luer lock adapter connected to a HTC PAL autosampler syringe. As the autosampler drew sample solution, analytes were extracted into the lumen of the extraction probe and transferred to a LC-MS system for further analysis. Various parameters affecting extraction efficacy were investigated including syringe fill strokes, syringe pull up volume, pull up delay and volume in the sample vial. The system was optimized for soft extraction of analytes and high sample throughput. Further, it was demonstrated that by flushing the EME-syringe with acidic wash buffer and reverting the applied electric potential, carry-over between samples can be reduced to below 1%. Performance of the system was characterized (RSD, <10%; R(2), 0.994) and finally, the EME-autosampler was used to analyze in vitro conversion of methadone into its main metabolite by rat liver microsomes and for demonstrating the potential of known CYP3A4 inhibitors to prevent metabolism of methadone. By making use of the high extraction speed of EME, a complete analytical workflow of purification, separation, and analysis of sample could be achieved within only 5.5 min. With the developed system large sequences of samples could be analyzed in a completely automated manner. This high degree of automation makes the developed EME-autosampler a powerful tool for a wide range of applications where high-throughput extractions are required before sample analysis.

  14. Active learning: a step towards automating medical concept extraction.

    PubMed

    Kholghi, Mahnoosh; Sitbon, Laurianne; Zuccon, Guido; Nguyen, Anthony

    2016-03-01

    This paper presents an automatic, active learning-based system for the extraction of medical concepts from clinical free-text reports. Specifically, (1) the contribution of active learning in reducing the annotation effort and (2) the robustness of incremental active learning framework across different selection criteria and data sets are determined. The comparative performance of an active learning framework and a fully supervised approach were investigated to study how active learning reduces the annotation effort while achieving the same effectiveness as a supervised approach. Conditional random fields as the supervised method, and least confidence and information density as 2 selection criteria for active learning framework were used. The effect of incremental learning vs standard learning on the robustness of the models within the active learning framework with different selection criteria was also investigated. The following 2 clinical data sets were used for evaluation: the Informatics for Integrating Biology and the Bedside/Veteran Affairs (i2b2/VA) 2010 natural language processing challenge and the Shared Annotated Resources/Conference and Labs of the Evaluation Forum (ShARe/CLEF) 2013 eHealth Evaluation Lab. The annotation effort saved by active learning to achieve the same effectiveness as supervised learning is up to 77%, 57%, and 46% of the total number of sequences, tokens, and concepts, respectively. Compared with the random sampling baseline, the saving is at least doubled. Incremental active learning is a promising approach for building effective and robust medical concept extraction models while significantly reducing the burden of manual annotation. © The Author 2015. Published by Oxford University Press on behalf of the American Medical Informatics Association. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  15. An automated approach for extracting Barrier Island morphology from digital elevation models

    NASA Astrophysics Data System (ADS)

    Wernette, Phillipe; Houser, Chris; Bishop, Michael P.

    2016-06-01

    The response and recovery of a barrier island to extreme storms depends on the elevation of the dune base and crest, both of which can vary considerably alongshore and through time. Quantifying the response to and recovery from storms requires that we can first identify and differentiate the dune(s) from the beach and back-barrier, which in turn depends on accurate identification and delineation of the dune toe, crest and heel. The purpose of this paper is to introduce a multi-scale automated approach for extracting beach, dune (dune toe, dune crest and dune heel), and barrier island morphology. The automated approach introduced here extracts the shoreline and back-barrier shoreline based on elevation thresholds, and extracts the dune toe, dune crest and dune heel based on the average relative relief (RR) across multiple spatial scales of analysis. The multi-scale automated RR approach to extracting dune toe, dune crest, and dune heel based upon relative relief is more objective than traditional approaches because every pixel is analyzed across multiple computational scales and the identification of features is based on the calculated RR values. The RR approach out-performed contemporary approaches and represents a fast objective means to define important beach and dune features for predicting barrier island response to storms. The RR method also does not require that the dune toe, crest, or heel are spatially continuous, which is important because dune morphology is likely naturally variable alongshore.

  16. Automated extraction and labelling of the arterial tree from whole-body MRA data.

    PubMed

    Shahzad, Rahil; Dzyubachyk, Oleh; Staring, Marius; Kullberg, Joel; Johansson, Lars; Ahlström, Håkan; Lelieveldt, Boudewijn P F; van der Geest, Rob J

    2015-08-01

    In this work, we present a fully automated algorithm for extraction of the 3D arterial tree and labelling the tree segments from whole-body magnetic resonance angiography (WB-MRA) sequences. The algorithm developed consists of two core parts (i) 3D volume reconstruction from different stations with simultaneous correction of different types of intensity inhomogeneity, and (ii) Extraction of the arterial tree and subsequent labelling of the pruned extracted tree. Extraction of the arterial tree is performed using the probability map of the "contrast" class, which is obtained as one of the results of the inhomogeneity correction scheme. We demonstrate that such approach is more robust than using the difference between the pre- and post-contrast channels traditionally used for this purpose. Labelling the extracted tree is performed by using a combination of graph-based and atlas-based approaches. Validation of our method with respect to the extracted tree was performed on the arterial tree subdivided into 32 segments, 82.4% of which were completely detected, 11.7% partially detected, and 5.9% were missed on a cohort of 35 subjects. With respect to automated labelling accuracy of the 32 segments, various registration strategies were investigated on a training set consisting of 10 scans. Further analysis on the test set consisting of 25 data sets indicates that 69% of the vessel centerline tree in the head and neck region, 80% in the thorax and abdomen region, and 84% in the legs was accurately labelled to the correct vessel segment. These results indicate clinical potential of our approach in enabling fully automated and accurate analysis of the entire arterial tree. This is the first study that not only automatically extracts the WB-MRA arterial tree, but also labels the vessel tree segments.

  17. Comparisons of Three Automated Systems for Genomic DNA Extraction in a Clinical Diagnostic Laboratory

    PubMed Central

    Lee, Jong-Han; Park, Yongjung; Choi, Jong Rak; Lee, Eun Kyung

    2010-01-01

    Purpose The extraction of nucleic acid is initially a limiting step for successful molecular-based diagnostic workup. This study aims to compare the effectiveness of three automated DNA extraction systems for clinical laboratory use. Materials and Methods Venous blood samples from 22 healthy volunteers were analyzed using QIAamp® Blood Mini Kit (Qiagen), MagNA Pure LC Nucleic Acid Isolation Kit I (Roche), and Magtration-Magnazorb DNA common kit-200N (PSS). The concentration of extracted DNAs was measured by NanoDrop ND-1000 (PeqLab). Also, extracted DNAs were confirmed by applying in direct agarose gel electrophoresis and were amplified by polymerase chain reaction (PCR) for human beta-globin gene. Results The corrected concentrations of extracted DNAs were 25.42 ± 8.82 ng/µL (13.49-52.85 ng/µL) by QIAamp® Blood Mini Kit (Qiagen), and 22.65 ± 14.49 ng/µL (19.18-93.39 ng/µL) by MagNA Pure LC Nucleic Acid Isolation Kit I, and 22.35 ± 6.47 ng/µL (12.57-35.08 ng/µL) by Magtration-Magnazorb DNA common kit-200N (PSS). No statistically significant difference was noticed among the three commercial kits (p > 0.05). Only the mean value of DNA purity through PSS was slightly lower than others. All the extracted DNAs were successfully identified in direct agarose gel electrophoresis. And all the product of beta-globin gene PCR showed a reproducible pattern of bands. Conclusion The effectiveness of the three automated extraction systems is of an equivalent level and good enough to produce reasonable results. Each laboratory could select the automated system according to its clinical and laboratory conditions. PMID:20046522

  18. AutoMate Express™ forensic DNA extraction system for the extraction of genomic DNA from biological samples.

    PubMed

    Liu, Jason Y; Zhong, Chang; Holt, Allison; Lagace, Robert; Harrold, Michael; Dixon, Alan B; Brevnov, Maxim G; Shewale, Jaiprakash G; Hennessy, Lori K

    2012-07-01

    The AutoMate Express™ Forensic DNA Extraction System was developed for automatic isolation of DNA from a variety of forensic biological samples. The performance of the system was investigated using a wide range of biological samples. Depending on the sample type, either PrepFiler™ lysis buffer or PrepFiler BTA™ lysis buffer was used to lyse the samples. After lysis and removal of the substrate using LySep™ column, the lysate in the sample tubes were loaded onto AutoMate Express™ instrument and DNA was extracted using one of the two instrument extraction protocols. Our study showed that DNA was recovered from as little as 0.025 μL of blood. DNA extracted from casework-type samples was free of detectable PCR inhibitors and the short tandem repeat profiles were complete, conclusive, and devoid of any PCR artifacts. The system also showed consistent performance from day-to-day operation. 2012 American Academy of Forensic Sciences. Published 2012. This article is a U.S. Government work and is in the public domain in the U.S.A.

  19. Automated segmentation and feature extraction of product inspection items

    NASA Astrophysics Data System (ADS)

    Talukder, Ashit; Casasent, David P.

    1997-03-01

    X-ray film and linescan images of pistachio nuts on conveyor trays for product inspection are considered. The final objective is the categorization of pistachios into good, blemished and infested nuts. A crucial step before classification is the separation of touching products and the extraction of features essential for classification. This paper addresses new detection and segmentation algorithms to isolate touching or overlapping items. These algorithms employ a new filter, a new watershed algorithm, and morphological processing to produce nutmeat-only images. Tests on a large database of x-ray film and real-time x-ray linescan images of around 2900 small, medium and large nuts showed excellent segmentation results. A new technique to detect and segment dark regions in nutmeat images is also presented and tested on approximately 300 x-ray film and approximately 300 real-time linescan x-ray images with 95-97 percent detection and correct segmentation. New algorithms are described that determine nutmeat fill ratio and locate splits in nutmeat. The techniques formulated in this paper are of general use in many different product inspection and computer vision problems.

  20. Comparative evaluation of automated and manual commercial DNA extraction methods for detection of Francisella tularensis DNA from suspensions and spiked swabs by real-time polymerase chain reaction.

    PubMed

    Dauphin, Leslie A; Walker, Roblena E; Petersen, Jeannine M; Bowen, Michael D

    2011-07-01

    This study evaluated commercial automated and manual DNA extraction methods for the isolation of Francisella tularensis DNA suitable for real-time polymerase chain reaction (PCR) analysis from cell suspensions and spiked cotton, foam, and polyester swabs. Two automated methods, the MagNA Pure Compact and the QIAcube, were compared to 4 manual methods, the IT 1-2-3 DNA sample purification kit, the MasterPure Complete DNA and RNA purification kit, the QIAamp DNA blood mini kit, and the UltraClean Microbial DNA isolation kit. The methods were compared using 6 F. tularensis strains representing the 2 subspecies which cause the majority of reported cases of tularemia in humans. Cell viability testing of the DNA extracts showed that all 6 extraction methods efficiently inactivated F. tularensis at concentrations of ≤10⁶ CFU/mL. Real-time PCR analysis using a multitarget 5' nuclease assay for F. tularensis revealed that the PCR sensitivity was equivalent using DNA extracted by the 2 automated methods and the manual MasterPure and QIAamp methods. These 4 methods resulted in significantly better levels of detection from bacterial suspensions and performed equivalently for spiked swab samples than the remaining 2. This study identifies optimal DNA extraction methods for processing swab specimens for the subsequent detection of F. tularensis DNA using real-time PCR assays. Furthermore, the results provide diagnostic laboratories with the option to select from 2 automated DNA extraction methods as suitable alternatives to manual methods for the isolation of DNA from F. tularensis.

  1. A fully automated liquid–liquid extraction system utilizing interface detection

    PubMed Central

    Maslana, Eugene; Schmitt, Robert; Pan, Jeffrey

    2000-01-01

    The development of the Abbott Liquid-Liquid Extraction Station was a result of the need for an automated system to perform aqueous extraction on large sets of newly synthesized organic compounds used for drug discovery. The system utilizes a cylindrical laboratory robot to shuttle sample vials between two loading racks, two identical extraction stations, and a centrifuge. Extraction is performed by detecting the phase interface (by difference in refractive index) of the moving column of fluid drawn from the bottom of each vial containing a biphasic mixture. The integration of interface detection with fluid extraction maximizes sample throughput. Abbott-developed electronics process the detector signals. Sample mixing is performed by high-speed solvent injection. Centrifuging of the samples reduces interface emulsions. Operating software permits the user to program wash protocols with any one of six solvents per wash cycle with as many cycle repeats as necessary. Station capacity is eighty, 15 ml vials. This system has proven successful with a broad spectrum of both ethyl acetate and methylene chloride based chemistries. The development and characterization of this automated extraction system will be presented. PMID:18924693

  2. Using a commercial DNA extraction kit to obtain RNA from mature rice kernels

    USDA-ARS?s Scientific Manuscript database

    Few RNA extraction protocols or commercial kits work well with the starchy endosperm of cereal grains. Standard RNA extraction protocols are time consuming, use large amounts of expensive chemicals, and leave behind hazardous wastes. However, there are numerous commercial DNA extraction kits that ...

  3. Automated renal histopathology: digital extraction and quantification of renal pathology

    NASA Astrophysics Data System (ADS)

    Sarder, Pinaki; Ginley, Brandon; Tomaszewski, John E.

    2016-03-01

    The branch of pathology concerned with excess blood serum proteins being excreted in the urine pays particular attention to the glomerulus, a small intertwined bunch of capillaries located at the beginning of the nephron. Normal glomeruli allow moderate amount of blood proteins to be filtered; proteinuric glomeruli allow large amount of blood proteins to be filtered. Diagnosis of proteinuric diseases requires time intensive manual examination of the structural compartments of the glomerulus from renal biopsies. Pathological examination includes cellularity of individual compartments, Bowman's and luminal space segmentation, cellular morphology, glomerular volume, capillary morphology, and more. Long examination times may lead to increased diagnosis time and/or lead to reduced precision of the diagnostic process. Automatic quantification holds strong potential to reduce renal diagnostic time. We have developed a computational pipeline capable of automatically segmenting relevant features from renal biopsies. Our method first segments glomerular compartments from renal biopsies by isolating regions with high nuclear density. Gabor texture segmentation is used to accurately define glomerular boundaries. Bowman's and luminal spaces are segmented using morphological operators. Nuclei structures are segmented using color deconvolution, morphological processing, and bottleneck detection. Average computation time of feature extraction for a typical biopsy, comprising of ~12 glomeruli, is ˜69 s using an Intel(R) Core(TM) i7-4790 CPU, and is ~65X faster than manual processing. Using images from rat renal tissue samples, automatic glomerular structural feature estimation was reproducibly demonstrated for 15 biopsy images, which contained 148 individual glomeruli images. The proposed method holds immense potential to enhance information available while making clinical diagnoses.

  4. Automated DNA extraction from genetically modified maize using aminosilane-modified bacterial magnetic particles.

    PubMed

    Ota, Hiroyuki; Lim, Tae-Kyu; Tanaka, Tsuyoshi; Yoshino, Tomoko; Harada, Manabu; Matsunaga, Tadashi

    2006-09-18

    A novel, automated system, PNE-1080, equipped with eight automated pestle units and a spectrophotometer was developed for genomic DNA extraction from maize using aminosilane-modified bacterial magnetic particles (BMPs). The use of aminosilane-modified BMPs allowed highly accurate DNA recovery. The (A(260)-A(320)):(A(280)-A(320)) ratio of the extracted DNA was 1.9+/-0.1. The DNA quality was sufficiently pure for PCR analysis. The PNE-1080 offered rapid assay completion (30 min) with high accuracy. Furthermore, the results of real-time PCR confirmed that our proposed method permitted the accurate determination of genetically modified DNA composition and correlated well with results obtained by conventional cetyltrimethylammonium bromide (CTAB)-based methods.

  5. Extraction of Prostatic Lumina and Automated Recognition for Prostatic Calculus Image Using PCA-SVM

    PubMed Central

    Wang, Zhuocai; Xu, Xiangmin; Ding, Xiaojun; Xiao, Hui; Huang, Yusheng; Liu, Jian; Xing, Xiaofen; Wang, Hua; Liao, D. Joshua

    2011-01-01

    Identification of prostatic calculi is an important basis for determining the tissue origin. Computation-assistant diagnosis of prostatic calculi may have promising potential but is currently still less studied. We studied the extraction of prostatic lumina and automated recognition for calculus images. Extraction of lumina from prostate histology images was based on local entropy and Otsu threshold recognition using PCA-SVM and based on the texture features of prostatic calculus. The SVM classifier showed an average time 0.1432 second, an average training accuracy of 100%, an average test accuracy of 93.12%, a sensitivity of 87.74%, and a specificity of 94.82%. We concluded that the algorithm, based on texture features and PCA-SVM, can recognize the concentric structure and visualized features easily. Therefore, this method is effective for the automated recognition of prostatic calculi. PMID:21461364

  6. Extraction of prostatic lumina and automated recognition for prostatic calculus image using PCA-SVM.

    PubMed

    Wang, Zhuocai; Xu, Xiangmin; Ding, Xiaojun; Xiao, Hui; Huang, Yusheng; Liu, Jian; Xing, Xiaofen; Wang, Hua; Liao, D Joshua

    2011-01-01

    Identification of prostatic calculi is an important basis for determining the tissue origin. Computation-assistant diagnosis of prostatic calculi may have promising potential but is currently still less studied. We studied the extraction of prostatic lumina and automated recognition for calculus images. Extraction of lumina from prostate histology images was based on local entropy and Otsu threshold recognition using PCA-SVM and based on the texture features of prostatic calculus. The SVM classifier showed an average time 0.1432 second, an average training accuracy of 100%, an average test accuracy of 93.12%, a sensitivity of 87.74%, and a specificity of 94.82%. We concluded that the algorithm, based on texture features and PCA-SVM, can recognize the concentric structure and visualized features easily. Therefore, this method is effective for the automated recognition of prostatic calculi.

  7. An integrated approach for automating validation of extracted ion chromatographic peaks.

    PubMed

    Nelson, William D; Viele, Kert; Lynn, Bert C

    2008-09-15

    Accurate determination of extracted ion chromatographic peak areas in isotope-labeled quantitative proteomics is difficult to automate. Manual validation of identified peaks is typically required. We have integrated a peak confidence scoring algorithm into existing tools which are compatible with analysis pipelines based on the standards from the Institute for Systems Biology. This algorithm automatically excludes incorrectly identified peaks, improving the accuracy of the final protein expression ratio calculation. http://www.chem.uky.edu/research/lynn/Nelson.pdf.

  8. Evaluation of an automated protocol for efficient and reliable DNA extraction of dietary samples.

    PubMed

    Wallinger, Corinna; Staudacher, Karin; Sint, Daniela; Thalinger, Bettina; Oehm, Johannes; Juen, Anita; Traugott, Michael

    2017-08-01

    Molecular techniques have become an important tool to empirically assess feeding interactions. The increased usage of next-generation sequencing approaches has stressed the need of fast DNA extraction that does not compromise DNA quality. Dietary samples here pose a particular challenge, as these demand high-quality DNA extraction procedures for obtaining the minute quantities of short-fragmented food DNA. Automatic high-throughput procedures significantly decrease time and costs and allow for standardization of extracting total DNA. However, these approaches have not yet been evaluated for dietary samples. We tested the efficiency of an automatic DNA extraction platform and a traditional CTAB protocol, employing a variety of dietary samples including invertebrate whole-body extracts as well as invertebrate and vertebrate gut content samples and feces. Extraction efficacy was quantified using the proportions of successful PCR amplifications of both total and prey DNA, and cost was estimated in terms of time and material expense. For extraction of total DNA, the automated platform performed better for both invertebrate and vertebrate samples. This was also true for prey detection in vertebrate samples. For the dietary analysis in invertebrates, there is still room for improvement when using the high-throughput system for optimal DNA yields. Overall, the automated DNA extraction system turned out as a promising alternative to labor-intensive, low-throughput manual extraction methods such as CTAB. It is opening up the opportunity for an extensive use of this cost-efficient and innovative methodology at low contamination risk also in trophic ecology.

  9. Rnnotator: an automated de novo transcriptome assembly pipeline from stranded RNA-Seq reads

    SciTech Connect

    Martin, Jeffrey; Bruno, Vincent M.; Fang, Zhide; Meng, Xiandong; Blow, Matthew; Zhang, Tao; Sherlock, Gavin; Snyder, Michael; Wang, Zhong

    2010-11-19

    Background: Comprehensive annotation and quantification of transcriptomes are outstanding problems in functional genomics. While high throughput mRNA sequencing (RNA-Seq) has emerged as a powerful tool for addressing these problems, its success is dependent upon the availability and quality of reference genome sequences, thus limiting the organisms to which it can be applied. Results: Here, we describe Rnnotator, an automated software pipeline that generates transcript models by de novo assembly of RNA-Seq data without the need for a reference genome. We have applied the Rnnotator assembly pipeline to two yeast transcriptomes and compared the results to the reference gene catalogs of these organisms. The contigs produced by Rnnotator are highly accurate (95percent) and reconstruct full-length genes for the majority of the existing gene models (54.3percent). Furthermore, our analyses revealed many novel transcribed regions that are absent from well annotated genomes, suggesting Rnnotator serves as a complementary approach to analysis based on a reference genome for comprehensive transcriptomics. Conclusions: These results demonstrate that the Rnnotator pipeline is able to reconstruct full-length transcripts in the absence of a complete reference genome.

  10. The Presence of Humic Substances and DNA in RNA Extracts Affects Hybridization Results

    PubMed Central

    Alm, Elizabeth Wheeler; Zheng, Dandan; Raskin, Lutgarde

    2000-01-01

    RNA extracts obtained from environmental samples are frequently contaminated with coextracted humic substances and DNA. It was demonstrated that the response in rRNA-targeted oligonucleotide probe hybridizations decreased as the concentrations of humic substances and DNA in RNA extracts increased. The decrease in hybridization signal in the presence of humic substances appeared to be due to saturation of the hybridization membrane with humic substances, resulting in a lower amount of target rRNA bound to the membrane. The decrease in hybridization response in the presence of low amounts of DNA may be the result of reduced rRNA target accessibility. The presence of high amounts of DNA in RNA extracts resulted in membrane saturation. Consistent with the observations for DNA contamination, the addition of poly(A) to RNA extracts, a common practice used to prepare RNA dilutions for membrane blotting, also reduced hybridization signals, likely because of reduced target accessibility and membrane saturation effects. PMID:11010915

  11. Application and evaluation of automated methods to extract neuroanatomical connectivity statements from free text.

    PubMed

    French, Leon; Lane, Suzanne; Xu, Lydia; Siu, Celia; Kwok, Cathy; Chen, Yiqi; Krebs, Claudia; Pavlidis, Paul

    2012-11-15

    Automated annotation of neuroanatomical connectivity statements from the neuroscience literature would enable accessible and large-scale connectivity resources. Unfortunately, the connectivity findings are not formally encoded and occur as natural language text. This hinders aggregation, indexing, searching and integration of the reports. We annotated a set of 1377 abstracts for connectivity relations to facilitate automated extraction of connectivity relationships from neuroscience literature. We tested several baseline measures based on co-occurrence and lexical rules. We compare results from seven machine learning methods adapted from the protein interaction extraction domain that employ part-of-speech, dependency and syntax features. Co-occurrence based methods provided high recall with weak precision. The shallow linguistic kernel recalled 70.1% of the sentence-level connectivity statements at 50.3% precision. Owing to its speed and simplicity, we applied the shallow linguistic kernel to a large set of new abstracts. To evaluate the results, we compared 2688 extracted connections with the Brain Architecture Management System (an existing database of rat connectivity). The extracted connections were connected in the Brain Architecture Management System at a rate of 63.5%, compared with 51.1% for co-occurring brain region pairs. We found that precision increases with the recency and frequency of the extracted relationships. The source code, evaluations, documentation and other supplementary materials are available at http://www.chibi.ubc.ca/WhiteText. paul@chibi.ubc.ca. Supplementary data are available at Bioinformatics Online.

  12. An advanced distributed automated extraction of drainage network model on high-resolution DEM

    NASA Astrophysics Data System (ADS)

    Mao, Y.; Ye, A.; Xu, J.; Ma, F.; Deng, X.; Miao, C.; Gong, W.; Di, Z.

    2014-07-01

    A high-resolution and high-accuracy drainage network map is a prerequisite for simulating the water cycle in land surface hydrological models. The objective of this study was to develop a new automated extraction of drainage network model, which can get high-precision continuous drainage network on high-resolution DEM (Digital Elevation Model). The high-resolution DEM need too much computer resources to extract drainage network. The conventional GIS method often can not complete to calculate on high-resolution DEM of big basins, because the number of grids is too large. In order to decrease the computation time, an advanced distributed automated extraction of drainage network model (Adam) was proposed in the study. The Adam model has two features: (1) searching upward from outlet of basin instead of sink filling, (2) dividing sub-basins on low-resolution DEM, and then extracting drainage network on sub-basins of high-resolution DEM. The case study used elevation data of the Shuttle Radar Topography Mission (SRTM) at 3 arc-second resolution in Zhujiang River basin, China. The results show Adam model can dramatically reduce the computation time. The extracting drainage network was continuous and more accurate than HydroSHEDS (Hydrological data and maps based on Shuttle Elevation Derivatives at multiple Scales).

  13. Automated information extraction of key trial design elements from clinical trial publications.

    PubMed

    de Bruijn, Berry; Carini, Simona; Kiritchenko, Svetlana; Martin, Joel; Sim, Ida

    2008-11-06

    Clinical trials are one of the most valuable sources of scientific evidence for improving the practice of medicine. The Trial Bank project aims to improve structured access to trial findings by including formalized trial information into a knowledge base. Manually extracting trial information from published articles is costly, but automated information extraction techniques can assist. The current study highlights a single architecture to extract a wide array of information elements from full-text publications of randomized clinical trials (RCTs). This architecture combines a text classifier with a weak regular expression matcher. We tested this two-stage architecture on 88 RCT reports from 5 leading medical journals, extracting 23 elements of key trial information such as eligibility rules, sample size, intervention, and outcome names. Results prove this to be a promising avenue to help critical appraisers, systematic reviewers, and curators quickly identify key information elements in published RCT articles.

  14. Comparison of QIAGEN automated nucleic acid extraction methods for CMV quantitative PCR testing.

    PubMed

    Miller, Steve; Seet, Henrietta; Khan, Yasmeen; Wright, Carolyn; Nadarajah, Rohan

    2010-04-01

    We examined the effect of nucleic acid extraction methods on the analytic characteristics of a quantitative polymerase chain reaction (PCR) assay for cytomegalovirus (CMV). Human serum samples were extracted with 2 automated instruments (BioRobot EZ1 and QIAsymphony SP, QIAGEN, Valencia, CA) and CMV PCR results compared with those of pp65 antigenemia testing. Both extraction methods yielded results that were comparably linear and precise, whereas the QIAsymphony SP had a slightly lower limit of detection (1.92 log(10) copies/mL vs 2.26 log(10) copies/mL). In both cases, PCR was more sensitive than CMV antigen detection, detecting CMV viremia in 12% (EZ1) and 21% (QIAsymphony) of antigen-negative specimens. This study demonstrates the feasibility of using 2 different extraction techniques to yield results within 0.5 log(10) copies/mL of the mean value, a level that would allow for clinical comparison between different laboratory assays.

  15. Bacterial and fungal DNA extraction from positive blood culture bottles: a manual and an automated protocol.

    PubMed

    Mäki, Minna

    2015-01-01

    When adapting a gene amplification-based method in a routine sepsis diagnostics using a blood culture sample as a specimen type, a prerequisite for a successful and sensitive downstream analysis is the efficient DNA extraction step. In recent years, a number of in-house and commercial DNA extraction solutions have become available. Careful evaluation in respect to cell wall disruption of various microbes and subsequent recovery of microbial DNA without putative gene amplification inhibitors should be conducted prior selecting the most feasible DNA extraction solution for the downstream analysis used. Since gene amplification technologies have been developed to be highly sensitive for a broad range of microbial species, it is also important to confirm that the used sample preparation reagents and materials are bioburden-free to avoid any risks for false-positive result reporting or interference of the diagnostic process. Here, one manual and one automated DNA extraction system feasible for blood culture samples are described.

  16. An automated approach to prepare tissue-derived spatially barcoded RNA-sequencing libraries

    PubMed Central

    Jemt, Anders; Salmén, Fredrik; Lundmark, Anna; Mollbrink, Annelie; Fernández Navarro, José; Ståhl, Patrik L.; Yucel-Lindberg, Tülay; Lundeberg, Joakim

    2016-01-01

    Sequencing the nucleic acid content of individual cells or specific biological samples is becoming increasingly common. This drives the need for robust, scalable and automated library preparation protocols. Furthermore, an increased understanding of tissue heterogeneity has lead to the development of several unique sequencing protocols that aim to retain or infer spatial context. In this study, a protocol for retaining spatial information of transcripts has been adapted to run on a robotic workstation. The method spatial transcriptomics is evaluated in terms of robustness and variability through the preparation of reference RNA, as well as through preparation and sequencing of six replicate sections of a gingival tissue biopsy from a patient with periodontitis. The results are reduced technical variability between replicates and a higher throughput, processing four times more samples with less than a third of the hands on time, compared to the standard protocol. PMID:27849009

  17. Improved protocol for high-quality co-extraction of DNA and RNA from rumen digesta.

    PubMed

    Popova, M; Martin, C; Morgavi, D P

    2010-07-01

    We report an improved method for total nucleic acids extraction from rumen content samples. The method employs bead beating, and phenol-chloroform extraction followed by saline-alcohol precipitation. Total nucleic acids and RNA yield and purity were assessed by spectrophotometric measurements; RNA integrity was estimated using Agilent RNA 6000 Nano Kit on an Agilent 2100 Bioanalyzer. The method provided total nucleic acids and RNA extracts of good quantity and quality. The extraction is not time consuming and it is valuable for ecological studies of rumen microbial community structure and gene expression.

  18. Improved RNA extraction and one-tube RT-PCR assay for simultaneous detection of control plant RNA plus several viruses in plant extracts.

    PubMed

    Nassuth, A; Pollari, E; Helmeczy, K; Stewart, S; Kofalvi, S A

    2000-10-01

    A procedure was developed for simultaneous detection of plant RNA viruses and of plant RNA, as a control. RT-PCR amplification with primers designed for the detection of the plant mRNAs encoding malate dehydrogenase (MDH) and the large subunit of ribulose bisphosphate carboxylase oxygenase (RubiscoL) was used for the development of a plant extraction procedure that consistently yields extracts that can be amplified. The control amplification was used successfully on extracts from cane, leaf and/or bud tissues from grapevine, apple, raspberry, strawberry, peach, apricot, plum and wheat. Multiplex RT-PCR conditions were established for the simultaneous detection in grapevine extracts of either arabis mosaic virus, rupestris stem pitting associated virus and malate dehydrogenase mRNA, or grapevine virus A, grapevine virus B, grapevine leafroll associated virus-3, and RubiscoL mRNA.

  19. Extraction of total RNA from Chrysanthemum containing high levels of phenolic and carbohydrates.

    PubMed

    Daohong, Wang; Bochu, Wang; Biao, Li; Chuanren, Duan; Jin, Zhang

    2004-07-15

    Some standard methods are available in total RNA extraction of plant tissue, which are effective for the ordinary plants. But it is difficult to extract the total RNA from the plants with high levels of phenolic compounds, carbohydrates, or other compounds that bind and/or coprecipitate with RNA. In this paper, a method was described that used the soluble polyvinylpyrrolidone (PVP), ethanol precipitation, phenol extraction and LiCl precipitation. By this method, RNA capable of reverse-transcription and polymerase chain reaction (PCR) amplification was isolated from Chrysanthemum that contains high levels of phenolic compounds and carbohydrates. The OD260/OD280 absorbance ratio is 2, and the RNA is intact. Other specialized RNA extraction methods failed to deliver suitable product. Bands of PCR products are clearly appearing on the polypropylene acyl gel electrophoresis (PAGE) gel, which indicates that the purity, concentration and integrity of total RNA were suitable for the sequent researches.

  20. PRECOG: a tool for automated extraction and visualization of fitness components in microbial growth phenomics.

    PubMed

    Fernandez-Ricaud, Luciano; Kourtchenko, Olga; Zackrisson, Martin; Warringer, Jonas; Blomberg, Anders

    2016-06-23

    Phenomics is a field in functional genomics that records variation in organismal phenotypes in the genetic, epigenetic or environmental context at a massive scale. For microbes, the key phenotype is the growth in population size because it contains information that is directly linked to fitness. Due to technical innovations and extensive automation our capacity to record complex and dynamic microbial growth data is rapidly outpacing our capacity to dissect and visualize this data and extract the fitness components it contains, hampering progress in all fields of microbiology. To automate visualization, analysis and exploration of complex and highly resolved microbial growth data as well as standardized extraction of the fitness components it contains, we developed the software PRECOG (PREsentation and Characterization Of Growth-data). PRECOG allows the user to quality control, interact with and evaluate microbial growth data with ease, speed and accuracy, also in cases of non-standard growth dynamics. Quality indices filter high- from low-quality growth experiments, reducing false positives. The pre-processing filters in PRECOG are computationally inexpensive and yet functionally comparable to more complex neural network procedures. We provide examples where data calibration, project design and feature extraction methodologies have a clear impact on the estimated growth traits, emphasising the need for proper standardization in data analysis. PRECOG is a tool that streamlines growth data pre-processing, phenotypic trait extraction, visualization, distribution and the creation of vast and informative phenomics databases.

  1. [Method for automated extraction and purification of nucleic acids and its implementation in microfluidic system].

    PubMed

    Mamaev, D D; Khodakov, D A; Dement'eva, E I; Filatov, I V; Iurasov, D A; Cherepanov, A I; Vasiliskov, V A; Smoldovskaia, O V; Zimenkov, D V; Griadunov, D A; Mikhaĭlovich, V M; Zasedatelev, A S

    2011-01-01

    A method and a microfluidic device for automated extraction and purification of nucleic acids from biological samples have been developed. The method involves disruption of bacterial cells and/or viral particles by combining enzymatic and chemical lysis procedures followed by solid-phase sorbent extraction and purification of nucleic acids. The procedure is carried out in an automated mode in a microfluidic module isolated from the outside environment, which minimizes contact of the researcher with potentially infectious samples and, consequently, decreases the risk of laboratory-acquired infections. The module includes reservoirs with lyophilized components for lysis and washing buffers; a microcolumn with a solid-phase sorbent; reservoirs containing water, ethanol, and water-ethanol buffer solutions for dissolving freeze-dried buffer components, rinsing the microcolumn, and eluting of nucleic acids; and microchannels and valves needed for directing fluids inside the module. The microfluidic module is placed into the control unit that delivers pressure, heats, mixes reagents, and flows solutions within the microfluidic module. The microfluidic system performs extraction and purification of nucleic acids with high efficiency in 40 min, and nucleic acids extracted can be directly used in PCR reaction and microarray assays.

  2. General principles and methods for routine automated microRNA in situ hybridization and double labeling with immunohistochemistry.

    PubMed

    Singh, U; Keirstead, N; Wolujczyk, A; Odin, M; Albassam, M; Garrido, R

    2014-05-01

    MicroRNAs (miRNAs) are small non-coding RNAs that modulate gene expression by binding to complementary sequences on target messenger RNA transcripts. Changes in the expression levels of specific miRNAs have been associated with a variety of disease conditions. We developed a reliable and high throughput in situ hybridization (ISH) method and optimized tissue fixation conditions for formalin fixed, paraffin embedded (FFPE) tissues. ISH methods were automated to visualize four miRNAs: miRNA-145 (smooth muscle cells), miRNA-126 (endothelial cells), miRNA-21 (neoplastic cells) and U6 small nuclear RNA (nuclear marker) using locked nucleic acid (LNA) probes and the Discovery Ultra Ventana(™) platform. The FFPE tissue sections were pretreated with protease 3, hybridized with probe concentrations of ≤ 25 nM; signal was detected using an enhanced, polymer-based detection method. The ISH signal was stronger and more uniform for tissue samples fixed for ≥ 48 h. To investigate the specificity of the method, we developed an automated dual ISH for miRNA-145 coupled with immunohistochemistry for smooth muscle actin, which confirmed the specific distribution of miRNA-145 to smooth muscle cells. These methods may be used routinely for exploratory studies of biomarker development, sample screening and understanding the role of miRNA in the pathophysiology of specific diseases.

  3. Brain MAPS: an automated, accurate and robust brain extraction technique using a template library

    PubMed Central

    Leung, Kelvin K.; Barnes, Josephine; Modat, Marc; Ridgway, Gerard R.; Bartlett, Jonathan W.; Fox, Nick C.; Ourselin, Sébastien

    2011-01-01

    Whole brain extraction is an important pre-processing step in neuro-image analysis. Manual or semi-automated brain delineations are labour-intensive and thus not desirable in large studies, meaning that automated techniques are preferable. The accuracy and robustness of automated methods are crucial because human expertise may be required to correct any sub-optimal results, which can be very time consuming. We compared the accuracy of four automated brain extraction methods: Brain Extraction Tool (BET), Brain Surface Extractor (BSE), Hybrid Watershed Algorithm (HWA) and a Multi-Atlas Propagation and Segmentation (MAPS) technique we have previously developed for hippocampal segmentation. The four methods were applied to extract whole brains from 682 1.5T and 157 3T T1-weighted MR baseline images from the Alzheimer’s Disease Neuroimaging Initiative database. Semi-automated brain segmentations with manual editing and checking were used as the gold-standard to compare with the results. The median Jaccard index of MAPS was higher than HWA, BET and BSE in 1.5T and 3T scans (p < 0.05, all tests), and the 1st-99th centile range of the Jaccard index of MAPS was smaller than HWA, BET and BSE in 1.5T and 3T scans (p < 0.05, all tests). HWA and MAPS were found to be best at including all brain tissues (median false negative rate ≤ 0.010% for 1.5T scans and ≤ 0.019% for 3T scans, both methods). The median Jaccard index of MAPS were similar in both 1.5T and 3T scans, whereas those of BET, BSE and HWA were higher in 1.5T scans than 3T scans (p < 0.05, all tests). We found that the diagnostic group had a small effect on the median Jaccard index of all four methods. In conclusion, MAPS had relatively high accuracy and low variability compared to HWA, BET and BSE in MR scans with and without atrophy. PMID:21195780

  4. Automated physics-based design of synthetic riboswitches from diverse RNA aptamers

    PubMed Central

    Espah Borujeni, Amin; Mishler, Dennis M.; Wang, Jingzhi; Huso, Walker; Salis, Howard M.

    2016-01-01

    Riboswitches are shape-changing regulatory RNAs that bind chemicals and regulate gene expression, directly coupling sensing to cellular actuation. However, it remains unclear how their sequence controls the physics of riboswitch switching and activation, particularly when changing the ligand-binding aptamer domain. We report the development of a statistical thermodynamic model that predicts the sequence-structure-function relationship for translation-regulating riboswitches that activate gene expression, characterized inside cells and within cell-free transcription–translation assays. Using the model, we carried out automated computational design of 62 synthetic riboswitches that used six different RNA aptamers to sense diverse chemicals (theophylline, tetramethylrosamine, fluoride, dopamine, thyroxine, 2,4-dinitrotoluene) and activated gene expression by up to 383-fold. The model explains how aptamer structure, ligand affinity, switching free energy and macromolecular crowding collectively control riboswitch activation. Our model-based approach for engineering riboswitches quantitatively confirms several physical mechanisms governing ligand-induced RNA shape-change and enables the development of cell-free and bacterial sensors for diverse applications. PMID:26621913

  5. Automated physics-based design of synthetic riboswitches from diverse RNA aptamers.

    PubMed

    Espah Borujeni, Amin; Mishler, Dennis M; Wang, Jingzhi; Huso, Walker; Salis, Howard M

    2016-01-08

    Riboswitches are shape-changing regulatory RNAs that bind chemicals and regulate gene expression, directly coupling sensing to cellular actuation. However, it remains unclear how their sequence controls the physics of riboswitch switching and activation, particularly when changing the ligand-binding aptamer domain. We report the development of a statistical thermodynamic model that predicts the sequence-structure-function relationship for translation-regulating riboswitches that activate gene expression, characterized inside cells and within cell-free transcription-translation assays. Using the model, we carried out automated computational design of 62 synthetic riboswitches that used six different RNA aptamers to sense diverse chemicals (theophylline, tetramethylrosamine, fluoride, dopamine, thyroxine, 2,4-dinitrotoluene) and activated gene expression by up to 383-fold. The model explains how aptamer structure, ligand affinity, switching free energy and macromolecular crowding collectively control riboswitch activation. Our model-based approach for engineering riboswitches quantitatively confirms several physical mechanisms governing ligand-induced RNA shape-change and enables the development of cell-free and bacterial sensors for diverse applications.

  6. Automated extraction of single H atoms with STM: tip state dependency.

    PubMed

    Møller, Morten; Jarvis, Samuel P; Guérinet, Laurent; Sharp, Peter; Woolley, Richard; Rahe, Philipp; Moriarty, Philip

    2017-02-17

    The atomistic structure of the tip apex plays a crucial role in performing reliable atomic-scale surface and adsorbate manipulation using scanning probe techniques. We have developed an automated extraction routine for controlled removal of single hydrogen atoms from the H:Si(100) surface. The set of atomic extraction protocols detect a variety of desorption events during scanning tunneling microscope (STM)-induced modification of the hydrogen-passivated surface. The influence of the tip state on the probability for hydrogen removal was examined by comparing the desorption efficiency for various classifications of STM topographs (rows, dimers, atoms, etc). We find that dimer-row-resolving tip apices extract hydrogen atoms most readily and reliably (and with least spurious desorption), while tip states which provide atomic resolution counter-intuitively have a lower probability for single H atom removal.

  7. Automated extraction of single H atoms with STM: tip state dependency

    NASA Astrophysics Data System (ADS)

    Møller, Morten; Jarvis, Samuel P.; Guérinet, Laurent; Sharp, Peter; Woolley, Richard; Rahe, Philipp; Moriarty, Philip

    2017-02-01

    The atomistic structure of the tip apex plays a crucial role in performing reliable atomic-scale surface and adsorbate manipulation using scanning probe techniques. We have developed an automated extraction routine for controlled removal of single hydrogen atoms from the H:Si(100) surface. The set of atomic extraction protocols detect a variety of desorption events during scanning tunneling microscope (STM)-induced modification of the hydrogen-passivated surface. The influence of the tip state on the probability for hydrogen removal was examined by comparing the desorption efficiency for various classifications of STM topographs (rows, dimers, atoms, etc). We find that dimer-row-resolving tip apices extract hydrogen atoms most readily and reliably (and with least spurious desorption), while tip states which provide atomic resolution counter-intuitively have a lower probability for single H atom removal.

  8. Factors controlling the manual and automated extraction of image information using imaging polarimetry

    NASA Astrophysics Data System (ADS)

    Duggin, Michael J.

    2004-07-01

    The factors governing the extraction of useful information from polarimetric images depend upon the image acquisition and analytical methodologies being used, and upon systematic and environmental variations present during the acquisition process. The acquisition process generally occurs with foreknowledge of the analysis to be used. Broadly, interactive image analysis and automated image analysis are two different procedures: in each case, there are technical challenges. Imaging polarimetry is more complex than other imaging methodologies, and produces an increased dimensionality. However, there are several potential broad areas of interactive (manual) and automated remote sensing in which imaging polarimetry can provide useful additional information. A review is presented of the factors controlling feature discrimination, of metrics that are used, and of some proposed directions for future research.

  9. Rapid and specific detection of tdh, trh1, and trh2 mRNA of Vibrio parahaemolyticus by transcription-reverse transcription concerted reaction with an automated system.

    PubMed

    Nakaguchi, Yoshitsugu; Ishizuka, Tetsuya; Ohnaka, Satoru; Hayashi, Toshinori; Yasukawa, Kiyoshi; Ishiguro, Takahiko; Nishibuchi, Mitsuaki

    2004-09-01

    Vibrio parahaemolyticus strains carrying the thermostable direct hemolysin (TDH) tdh gene, the TDH-related hemolysin (trh) gene, or both genes are considered virulent strains. We previously demonstrated that the transcription-reverse transcription concerted (TRC) method could be used to quantify the amount of mRNA transcribed from the tdh gene by using an automated detection system. In this study, we devised two TRC-based assays to quantify the mRNAs transcribed from the trh1 and trh2 genes, the two representative trh genes. The TRC-based detection assays for the tdh, trh1, and trh2 transcripts could specifically and quantitatively detect 10(3) to 10(7) copies of the corresponding calibrator RNAs. We examined by the three TRC assays the total RNA preparations extracted from 103 strains of Vibrio parahaemolyticus carrying the tdh, trh1, or trh2 gene in various combinations. The tdh, trh1, and trh2 mRNAs in the total RNA preparations were specifically quantified, and the time needed for detection ranged from 9 to 19 min, from 14 to 18 min, and from 9 to 12 min, respectively. The results showed that this automated TRC assays could detect the tdh, trh1, and trh2 mRNAs specifically, quantitatively, and rapidly. The relative levels of TDH determined by the immunological method and that of tdh mRNA determined by the TRC assays for most tdh-positive strains correlated. Interestingly, the levels of TDH produced from the strains carrying both tdh and trh genes were lower than those carrying only the tdh gene, whereas the levels of mRNA did not significantly differ between the two groups.

  10. Automated Kinematic Extraction of Wing and Body Motions of Free Flying Diptera

    NASA Astrophysics Data System (ADS)

    Kostreski, Nicholas I.

    In the quest to understand the forces generated by micro aerial systems powered by oscillating appendages, it is necessary to study the kinematics that generate those forces. Automated and manual tracking techniques were developed to extract the complex wing and body motions of dipteran insects, ideal micro aerial systems, in free flight. Video sequences were captured by three high speed cameras (7500 fps) oriented orthogonally around a clear flight test chamber. Synchronization and image-based triggering were made possible by an automated triggering circuit. A multi-camera calibration was implemented using image-based tracking techniques. Three-dimensional reconstructions of the insect were generated from the 2-D images by shape from silhouette (SFS) methods. An intensity based segmentation of the wings and body was performed using a mixture of Gaussians. In addition to geometric and cost based filtering, spectral clustering was also used to refine the reconstruction and Principal Component Analysis (PCA) was performed to find the body roll axis and wing-span axes. The unobservable roll state of the cylindrically shaped body was successfully estimated by combining observations of the wing kinematics with a wing symmetry assumption. Wing pitch was determined by a ray tracing technique to compute and minimize a point-to-line cost function. Linear estimation with assumed motion models was accomplished by discrete Kalman filtering the measured body states. Generative models were developed for different species of diptera for model based tracking, simulation, and extraction of inertial properties. Manual and automated tracking results were analyzed and insect flight simulation videos were developed to quantify ground truth errors for an assumed model. The results demonstrated the automated tracker to have comparable performance to a human digitizer, though manual techniques displayed superiority during aggressive maneuvers and image blur. Both techniques demonstrated

  11. Toward high-throughput phenotyping: unbiased automated feature extraction and selection from knowledge sources.

    PubMed

    Yu, Sheng; Liao, Katherine P; Shaw, Stanley Y; Gainer, Vivian S; Churchill, Susanne E; Szolovits, Peter; Murphy, Shawn N; Kohane, Isaac S; Cai, Tianxi

    2015-09-01

    Analysis of narrative (text) data from electronic health records (EHRs) can improve population-scale phenotyping for clinical and genetic research. Currently, selection of text features for phenotyping algorithms is slow and laborious, requiring extensive and iterative involvement by domain experts. This paper introduces a method to develop phenotyping algorithms in an unbiased manner by automatically extracting and selecting informative features, which can be comparable to expert-curated ones in classification accuracy. Comprehensive medical concepts were collected from publicly available knowledge sources in an automated, unbiased fashion. Natural language processing (NLP) revealed the occurrence patterns of these concepts in EHR narrative notes, which enabled selection of informative features for phenotype classification. When combined with additional codified features, a penalized logistic regression model was trained to classify the target phenotype. The authors applied our method to develop algorithms to identify patients with rheumatoid arthritis and coronary artery disease cases among those with rheumatoid arthritis from a large multi-institutional EHR. The area under the receiver operating characteristic curves (AUC) for classifying RA and CAD using models trained with automated features were 0.951 and 0.929, respectively, compared to the AUCs of 0.938 and 0.929 by models trained with expert-curated features. Models trained with NLP text features selected through an unbiased, automated procedure achieved comparable or slightly higher accuracy than those trained with expert-curated features. The majority of the selected model features were interpretable. The proposed automated feature extraction method, generating highly accurate phenotyping algorithms with improved efficiency, is a significant step toward high-throughput phenotyping. © The Author 2015. Published by Oxford University Press on behalf of the American Medical Informatics Association. All

  12. Automated extraction of natural drainage density patterns for the conterminous United States through high performance computing

    USGS Publications Warehouse

    Stanislawski, Larry V.; Falgout, Jeff T.; Buttenfield, Barbara P.

    2015-01-01

    Hydrographic networks form an important data foundation for cartographic base mapping and for hydrologic analysis. Drainage density patterns for these networks can be derived to characterize local landscape, bedrock and climate conditions, and further inform hydrologic and geomorphological analysis by indicating areas where too few headwater channels have been extracted. But natural drainage density patterns are not consistently available in existing hydrographic data for the United States because compilation and capture criteria historically varied, along with climate, during the period of data collection over the various terrain types throughout the country. This paper demonstrates an automated workflow that is being tested in a high-performance computing environment by the U.S. Geological Survey (USGS) to map natural drainage density patterns at the 1:24,000-scale (24K) for the conterminous United States. Hydrographic network drainage patterns may be extracted from elevation data to guide corrections for existing hydrographic network data. The paper describes three stages in this workflow including data pre-processing, natural channel extraction, and generation of drainage density patterns from extracted channels. The workflow is concurrently implemented by executing procedures on multiple subbasin watersheds within the U.S. National Hydrography Dataset (NHD). Pre-processing defines parameters that are needed for the extraction process. Extraction proceeds in standard fashion: filling sinks, developing flow direction and weighted flow accumulation rasters. Drainage channels with assigned Strahler stream order are extracted within a subbasin and simplified. Drainage density patterns are then estimated with 100-meter resolution and subsequently smoothed with a low-pass filter. The extraction process is found to be of better quality in higher slope terrains. Concurrent processing through the high performance computing environment is shown to facilitate and refine

  13. [Automated RNA amplification for the rapid identification of Mycobacterium tuberculosis complex in respiratory specimens].

    PubMed

    Drouillon, V; Houriez, F; Buze, M; Lagrange, P; Herrmann, J-L

    2006-01-01

    Rapid and sensitive detection of Mycobacterium tuberculosis complex (MTB) directly on clinical respiratory specimens is essential for a correct management of patients suspected of tuberculosis. For this purpose PCR-based kits are available to detect MTB in respiratory specimen but most of them need at least 4 hours to be completed. New methods, based on TRC method (TRC: Transcription Reverse transcription Concerted--TRCRapid M. Tuberculosis--Tosoh Bioscience, Tokyo, Japon) and dedicated monitor have been developed. A new kit (TRC Rapid M. tuberculosis and Real-time monitor TRCRapid-160, Tosoh Corporation, Japan) enabling one step amplification and real-time detection of MTB 16S rRNA by a combination of intercalative dye oxazole yellow-linked DNA probe and isothermal RNA amplification directly on respiratory specimens has been tested in our laboratory. 319 respiratory specimens were tested in this preliminary study and results were compared to smear and culture. Fourteen had a positive culture for MTB. Among theses samples, smear was positive in 11 cases (78.6%) and TRC process was positive in 8 cases (57.1%). Overall sensitivity of TRC compared to smear positive samples is 73%. Theses first results demonstrated that a rapid identification of MTB was possible (less than 2 processing hours for 14 specimens and about 1 hour for 1 specimen) in most cases of smear positive samples using ready to use reagents for real time detection of MTB rRNA in clinical samples. New pretreatment and extraction reagents kits to increase the stability of the sputum RNA and the extraction efficiency are now tested in our laboratory.

  14. Splicing of arabidopsis tRNA(Met) precursors in tobacco cell and wheat germ extracts.

    PubMed

    Akama, K; Junker, V; Yukawa, Y; Sugiura, M; Beier, H

    2000-09-01

    Intron-containing tRNA genes are exceptional within nuclear plant genomes. It appears that merely two tRNA gene families coding for tRNA(GpsiA(Tyr)) and elongator tRNA(CmAU(Met)) contain intervening sequences. We have previously investigated the features required by wheat germ splicing endonuclease for efficient and accurate intron excision from Arabidopsis pre-tRNA(Tyr). Here we have studied the expression of an Arabidopsis elongator tRNA(Met) gene in two plant extracts of different origin. This gene was first transcribed either in HeLa or in tobacco cell nuclear extract and splicing of intron-containing tRNA(Met) precursors was then examined in wheat germ S23 extract and in the tobacco system. The results show that conversion of pre-tRNA(Met) to mature tRNA proceeds very efficiently in both plant extracts. In order to elucidate the potential role of specific nucleotides at the 3' and 5' splice sites and of a structured intron for pre-tRNA(Met) splicing in either extract, we have performed a systematic survey by mutational analyses. The results show that cytidine residues at intron-exon boundaries impair pre-tRNA(Met) splicing and that a highly structured intron is indispensable for pre-tRNA(Met) splicing. tRNA precursors with an extended anticodon stem of three to four base pairs are readily accepted as substrates by wheat and tobacco splicing endonuclease, whereas pre-tRNA molecules that can form an extended anticodon stem of only two putative base pairs are not spliced at all. An amber suppressor, generated from the intron-containing elongator tRNA(Met) gene, is efficiently processed and spliced in both plant extracts.

  15. Automated milk fat extraction for the analyses of persistent organic pollutants.

    PubMed

    Archer, Jeffrey C; Jenkins, Roy G

    2017-01-15

    We have utilized an automated acid hydrolysis technology, followed by an abbreviated Soxhlet extraction technique to obtain fat from whole milk for the determination of persistent organic pollutants, namely polychlorinated dibenzo-p-dioxins, polychlorinated dibenzofurans and polychlorinated biphenyls. The process simply involves (1) pouring the liquid milk into the hydrolysis beaker with reagents and standards, (2) drying the obtained fat on a filter paper and (3) obtaining pure fat via the modified Soxhlet extraction using 100mL of hexane per sample. This technique is in contrast to traditional manually intense liquid-liquid extractions and avoids the preparatory step of freeze-drying the samples for pressurized liquid extractions. Along with these extraction improvements, analytical results closely agree between the methods, thus no quality has been compromised. The native spike (n=12) and internal standard (n=24) precision and accuracy results are within EPA Methods 1613 and 1668 limits. While the median (n=6) Toxic Equivalency Quotient (TEQ) for polychlorinated dibenzo-p-dioxins/polychlorinated dibenzofurans and the concentration of the marker polychlorinated biphenyls show a percent difference of 1% and 12%, respectively, compared to 315 previously analyzed milk samples at the same laboratory using liquid-liquid extraction. During our feasibility studies, both egg and fish tissue show substantial promise using this technique as well.

  16. Comparison of manual and automated nucleic acid extraction methods from clinical specimens for microbial diagnosis purposes.

    PubMed

    Wozniak, Aniela; Geoffroy, Enrique; Miranda, Carolina; Castillo, Claudia; Sanhueza, Francia; García, Patricia

    2016-11-01

    The choice of nucleic acids (NAs) extraction method for molecular diagnosis in microbiology is of major importance because of the low microbial load, different nature of microorganisms, and clinical specimens. The NA yield of different extraction methods has been mostly studied using spiked samples. However, information from real human clinical specimens is scarce. The purpose of this study was to compare the performance of a manual low-cost extraction method (Qiagen kit or salting-out extraction method) with the automated high-cost MagNAPure Compact method. According to cycle threshold values for different pathogens, MagNAPure is as efficient as Qiagen for NA extraction from noncomplex clinical specimens (nasopharyngeal swab, skin swab, plasma, respiratory specimens). In contrast, according to cycle threshold values for RNAseP, MagNAPure method may not be an appropriate method for NA extraction from blood. We believe that MagNAPure versatility reduced risk of cross-contamination and reduced hands-on time compensates its high cost. Copyright © 2016 Elsevier Inc. All rights reserved.

  17. Comparative evaluation of different extraction and quantification methods for forensic RNA analysis.

    PubMed

    Grabmüller, Melanie; Madea, Burkhard; Courts, Cornelius

    2015-05-01

    Since about 2005, there is increasing interest in forensic RNA analysis whose versatility may very favorably complement traditional DNA profiling in forensic casework. There is, however, no method available specifically dedicated for extraction of RNA from forensically relevant sample material. In this study we compared five commercially available and commonly used RNA extraction kits and methods (mirVana™ miRNA Isolation Kit Ambion; Trizol® Reagent, Invitrogen; NucleoSpin® miRNA Kit Macherey-Nagel; AllPrep DNA/RNA Mini Kit and RNeasy® Mini Kit both Qiagen) to assess their relative effectiveness of yielding RNA of good quality and their compatibility with co-extraction of DNA amenable to STR profiling. We set up samples of small amounts of dried blood, liquid saliva, semen and buccal mucosa that were aged for different time intervals for co-extraction of RNA and DNA. RNA quality was assessed by determination of 'RNA integrity number' (RIN) and quantitative PCR based expression analysis. DNA quality was assessed via monitoring STR typing success rates. By comparison, the different methods exhibited considerable differences between RNA and DNA yields, RNA quality values and expression levels, and STR profiling success, with the AllPrep DNA/RNA Mini Kit and the NucleoSpin® miRNA Kit excelling at DNA co-extraction and RNA results, respectively. Overall, there was no 'best' method to satisfy all demands of comprehensible co-analysis of RNA and DNA and it appears that each method has specific merits and flaws. We recommend to cautiously choose from available methods and align its characteristics with the needs of the experimental setting at hand.

  18. Automated extraction of DNA from biological stains on fabric from crime cases. A comparison of a manual and three automated methods.

    PubMed

    Stangegaard, Michael; Hjort, Benjamin B; Hansen, Thomas N; Hoflund, Anders; Mogensen, Helle S; Hansen, Anders J; Morling, Niels

    2013-05-01

    The presence of PCR inhibitors in extracted DNA may interfere with the subsequent quantification and short tandem repeat (STR) reactions used in forensic genetic DNA typing. DNA extraction from fabric for forensic genetic purposes may be challenging due to the occasional presence of PCR inhibitors that may be co-extracted with the DNA. Using 120 forensic trace evidence samples consisting of various types of fabric, we compared three automated DNA extraction methods based on magnetic beads (PrepFiler Express Forensic DNA Extraction Kit on an AutoMate Express, QIAsyphony DNA Investigator kit either with the sample pre-treatment recommended by Qiagen or an in-house optimized sample pre-treatment on a QIAsymphony SP) and one manual method (Chelex) with the aim of reducing the amount of PCR inhibitors in the DNA extracts and increasing the proportion of reportable STR-profiles. A total of 480 samples were processed. The highest DNA recovery was obtained with the PrepFiler Express kit on an AutoMate Express while the lowest DNA recovery was obtained using a QIAsymphony SP with the sample pre-treatment recommended by Qiagen. Extraction using a QIAsymphony SP with the sample pre-treatment recommended by Qiagen resulted in the lowest percentage of PCR inhibition (0%) while extraction using manual Chelex resulted in the highest percentage of PCR inhibition (51%). The largest number of reportable STR-profiles was obtained with DNA from samples extracted with the PrepFiler Express kit (75%) while the lowest number was obtained with DNA from samples extracted using a QIAsymphony SP with the sample pre-treatment recommended by Qiagen (41%). Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  19. Establishing a novel automated magnetic bead-based method for the extraction of DNA from a variety of forensic samples.

    PubMed

    Witt, Sebastian; Neumann, Jan; Zierdt, Holger; Gébel, Gabriella; Röscheisen, Christiane

    2012-09-01

    Automated systems have been increasingly utilized for DNA extraction by many forensic laboratories to handle growing numbers of forensic casework samples while minimizing the risk of human errors and assuring high reproducibility. The step towards automation however is not easy: The automated extraction method has to be very versatile to reliably prepare high yields of pure genomic DNA from a broad variety of sample types on different carrier materials. To prevent possible cross-contamination of samples or the loss of DNA, the components of the kit have to be designed in a way that allows for the automated handling of the samples with no manual intervention necessary. DNA extraction using paramagnetic particles coated with a DNA-binding surface is predestined for an automated approach. For this study, we tested different DNA extraction kits using DNA-binding paramagnetic particles with regard to DNA yield and handling by a Freedom EVO(®)150 extraction robot (Tecan) equipped with a Te-MagS magnetic separator. Among others, the extraction kits tested were the ChargeSwitch(®)Forensic DNA Purification Kit (Invitrogen), the PrepFiler™Automated Forensic DNA Extraction Kit (Applied Biosystems) and NucleoMag™96 Trace (Macherey-Nagel). After an extensive test phase, we established a novel magnetic bead extraction method based upon the NucleoMag™ extraction kit (Macherey-Nagel). The new method is readily automatable and produces high yields of DNA from different sample types (blood, saliva, sperm, contact stains) on various substrates (filter paper, swabs, cigarette butts) with no evidence of a loss of magnetic beads or sample cross-contamination. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

  20. An integrated approach for automating validation of extracted ion chromatographic peaks

    PubMed Central

    Nelson, William D.; Viele, Kert; Lynn, Bert C.

    2008-01-01

    Summary: Accurate determination of extracted ion chromatographic peak areas in isotope-labeled quantitative proteomics is difficult to automate. Manual validation of identified peaks is typically required. We have integrated a peak confidence scoring algorithm into existing tools which are compatible with analysis pipelines based on the standards from the Institute for Systems Biology. This algorithm automatically excludes incorrectly identified peaks, improving the accuracy of the final protein expression ratio calculation. Contact: wnels2@uky.edu Source and Supplementary Information: http://www.chem.uky.edu/research/lynn/Nelson.pdf PMID:18653519

  1. Automated solid-phase extraction of herbicides from water for gas chromatographic-mass spectrometric analysis

    USGS Publications Warehouse

    Meyer, M.T.; Mills, M.S.; Thurman, E.M.

    1993-01-01

    An automated solid-phase extraction (SPE) method was developed for the pre-concentration of chloroacetanilide and triazine herbicides, and two triazine metabolites from 100-ml water samples. Breakthrough experiments for the C18 SPE cartridge show that the two triazine metabolites are not fully retained and that increasing flow-rate decreases their retention. Standard curve r2 values of 0.998-1.000 for each compound were consistently obtained and a quantitation level of 0.05 ??g/l was achieved for each compound tested. More than 10,000 surface and ground water samples have been analyzed by this method.

  2. Improved RNA extraction method using the BioMasher and BioMasher power-plus.

    PubMed

    Yamamoto, Takuji; Nakashima, Kentaro; Maruta, Yukio; Kiriyama, Tomomi; Sasaki, Michi; Sugiyama, Shunpei; Suzuki, Kana; Fujisaki, Hitomi; Sasaki, Jun; Kaku-Ushiki, Yuko; Tanida, Masatoshi; Irie, Shinkichi; Hattori, Shunji

    2012-12-01

    The BioMasher is a disposable homogenizer that was developed to homogenize bovine brain tissue for bovine spongiform encephalopathy diagnosis. Capable of preventing the biohazard risk from infectious samples, it also prevents cross-contamination among samples. The BioMasher is thus widely used in biochemical research, especially for RNA extraction. Here, we tested a novel BioMasher application for RNA extraction from animal and plant tissues. We also developed a grinding machine specific for the BioMasher, named the BioMasher Power-Plus. We developed RNA extraction protocols using the BioMasher combined with the BioMasher Power-Plus. We compared RNA extraction efficiency of the BioMasher with that of the FastPrep and the glass homogenizer. Though the RNA extraction efficiency by the BioMasher was nearly equivalent to that of the FastPrep and the glass homogenizer, sample preparation time was shorter for the BioMasher. The utility of RNA extraction by the BioMasher was examined in mouse, rat, and tomato tissue samples. In the rodent tissues, the highest extraction efficiency of total RNA was from liver, with lowest efficiency from fibrous tissues such as muscle. The quality of extracted total RNA was confirmed by agarose gel electrophoresis which produced highly visible clear bands of 18S and 28S rRNAs. Reproducibility among different operators in RNA extraction from tomato roots was improved by using the BioMasher Power-Plus. The BioMasher and BioMasher Power-Plus provide an effective and easy homogenization method for total RNA extraction from some rodent and plant tissues.

  3. Extraction of RNA from the plant Kalanchoë daigremontiana.

    PubMed

    Garcês, Helena; Sinha, Neelima

    2009-10-01

    This protocol describes how to isolate total RNA from several tissues of Kalanchoë daigremontiana. Total RNA can be isolated by using the TRI-reagent method, scaled up for processing 2 g of tissue, or by using the protocol described here, which gives higher concentrations of high quality RNA. The resulting RNA can be used for various applications including generation of cDNA for reverse transcriptase-PCR (RT-PCR), Northern blots, or other purposes.

  4. Application and evaluation of automated methods to extract neuroanatomical connectivity statements from free text

    PubMed Central

    Pavlidis, Paul

    2012-01-01

    Motivation: Automated annotation of neuroanatomical connectivity statements from the neuroscience literature would enable accessible and large-scale connectivity resources. Unfortunately, the connectivity findings are not formally encoded and occur as natural language text. This hinders aggregation, indexing, searching and integration of the reports. We annotated a set of 1377 abstracts for connectivity relations to facilitate automated extraction of connectivity relationships from neuroscience literature. We tested several baseline measures based on co-occurrence and lexical rules. We compare results from seven machine learning methods adapted from the protein interaction extraction domain that employ part-of-speech, dependency and syntax features. Results: Co-occurrence based methods provided high recall with weak precision. The shallow linguistic kernel recalled 70.1% of the sentence-level connectivity statements at 50.3% precision. Owing to its speed and simplicity, we applied the shallow linguistic kernel to a large set of new abstracts. To evaluate the results, we compared 2688 extracted connections with the Brain Architecture Management System (an existing database of rat connectivity). The extracted connections were connected in the Brain Architecture Management System at a rate of 63.5%, compared with 51.1% for co-occurring brain region pairs. We found that precision increases with the recency and frequency of the extracted relationships. Availability and implementation: The source code, evaluations, documentation and other supplementary materials are available at http://www.chibi.ubc.ca/WhiteText. Contact: paul@chibi.ubc.ca Supplementary information: Supplementary data are available at Bioinformatics Online. PMID:22954628

  5. Automated CO2 extraction from air for clumped isotope analysis in the atmo- and biosphere

    NASA Astrophysics Data System (ADS)

    Hofmann, Magdalena; Ziegler, Martin; Pons, Thijs; Lourens, Lucas; Röckmann, Thomas

    2015-04-01

    The conventional stable isotope ratios 13C/12C and 18O/16O in atmospheric CO2 are a powerful tool for unraveling the global carbon cycle. In recent years, it has been suggested that the abundance of the very rare isotopologue 13C18O16O on m/z 47 might be a promising tracer to complement conventional stable isotope analysis of atmospheric CO2 [Affek and Eiler, 2006; Affek et al. 2007; Eiler and Schauble, 2004; Yeung et al., 2009]. Here we present an automated analytical system that is designed for clumped isotope analysis of atmo- and biospheric CO2. The carbon dioxide gas is quantitatively extracted from about 1.5L of air (ATP). The automated stainless steel extraction and purification line consists of three main components: (i) a drying unit (a magnesium perchlorate unit and a cryogenic water trap), (ii) two CO2 traps cooled with liquid nitrogen [Werner et al., 2001] and (iii) a GC column packed with Porapak Q that can be cooled with liquid nitrogen to -30°C during purification and heated up to 230°C in-between two extraction runs. After CO2 extraction and purification, the CO2 is automatically transferred to the mass spectrometer. Mass spectrometric analysis of the 13C18O16O abundance is carried out in dual inlet mode on a MAT 253 mass spectrometer. Each analysis generally consists of 80 change-over-cycles. Three additional Faraday cups were added to the mass spectrometer for simultaneous analysis of the mass-to-charge ratios 44, 45, 46, 47, 48 and 49. The reproducibility for δ13C, δ18O and Δ47 for repeated CO2 extractions from air is in the range of 0.11o (SD), 0.18o (SD) and 0.02 (SD)o respectively. This automated CO2 extraction and purification system will be used to analyse the clumped isotopic signature in atmospheric CO2 (tall tower, Cabauw, Netherlands) and to study the clumped isotopic fractionation during photosynthesis (leaf chamber experiments) and soil respiration. References Affek, H. P., Xu, X. & Eiler, J. M., Geochim. Cosmochim. Acta 71, 5033

  6. Comparison of two methods for RNA extraction from the nucleus pulposus of intervertebral discs.

    PubMed

    Gan, M F; Yang, H L; Qian, J L; Wu, C S; Yuan, C X; Li, X F; Zou, J

    2016-06-03

    RNA extraction from the nucleus pulposus of intervertebral discs has been extensively used in orthopedic studies. We compared two methods for extracting RNA from the nucleus pulposus: liquid nitrogen grinding and enzyme digestion. The RNA was detected by agarose gel electrophoresis, and the purity was evaluated by absorbance ratio using a spectrophotometer. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression was assayed by reverse transcription-polymerase chain reaction (RT-PCR). Thirty human lumbar intervertebral discs were used in this study. The liquid nitrogen-grinding method was used for RNA extraction from 15 samples, and the mean RNA concentration was 491.04 ± 44.16 ng/mL. The enzyme digestion method was used on 15 samples, and the mean RNA concentration was 898.42 ± 38.64 ng/mL. The statistical analysis revealed that there was a significant difference in concentration between the different methods. Apparent 28S, 18S, and 5S bands were detectable in RNA extracted using the enzyme digestion method, whereas no 28S or 18S bands were detected in RNA extracted using the liquid nitrogen-grinding method. The GAPDH band was visible, and no non-specific band was detected in the RT-PCR assay by the enzyme digestion method. Therefore, the enzyme digestion method is an efficient and easy method for RNA extraction from the nucleus pulposus of intervertebral discs for further intervertebral disc degeneration-related studies.

  7. Integrated DNA and RNA extraction using magnetic beads from viral pathogens causing acute respiratory infections.

    PubMed

    He, Hui; Li, Rongqun; Chen, Yi; Pan, Ping; Tong, Wenjuan; Dong, Xueyan; Chen, Yueming; Yu, Daojun

    2017-03-23

    Current extraction methods often extract DNA and RNA separately, and few methods are capable of co-extracting DNA and RNA from sputum. We established a nucleic acid co-extraction method from sputum based on magnetic beads and optimized the method by evaluating influencing factors, such as the guanidinium thiocyanate (GTC) and dithiothreitol (DTT) concentrations, magnetic bead amount, incubation temperature, lysis buffer pH and RNA carrier type. The feasibility of the simultaneous nucleic acid co-extraction method was evaluated by amplifying DNA and RNA viruses from a single clinical specimen with a multiplex RT-qPCR method. Both DNA and RNA were most efficiently extracted when the GTC and DTT concentrations were 2.0 M and 80 mM, respectively, 20 μl magnetic beads were added, the incubation temperature was 80 °C, the pH was 8 or 9, and RNA carrier A was used. Therefore, we established a simple method to extract nucleic acids from two important respiratory viruses compared with other commercial kits. This magnetic beads-based co-extraction method for sputum followed by a multiplex RT-qPCR can rapidly and precisely detect DNA and RNA viruses from a single clinical specimen and has many advantages, such as decreased time, low cost, and a lack of harmful chemicals.

  8. Integrated DNA and RNA extraction using magnetic beads from viral pathogens causing acute respiratory infections

    PubMed Central

    He, Hui; Li, Rongqun; Chen, Yi; Pan, Ping; Tong, Wenjuan; Dong, Xueyan; Chen, Yueming; Yu, Daojun

    2017-01-01

    Current extraction methods often extract DNA and RNA separately, and few methods are capable of co-extracting DNA and RNA from sputum. We established a nucleic acid co-extraction method from sputum based on magnetic beads and optimized the method by evaluating influencing factors, such as the guanidinium thiocyanate (GTC) and dithiothreitol (DTT) concentrations, magnetic bead amount, incubation temperature, lysis buffer pH and RNA carrier type. The feasibility of the simultaneous nucleic acid co-extraction method was evaluated by amplifying DNA and RNA viruses from a single clinical specimen with a multiplex RT-qPCR method. Both DNA and RNA were most efficiently extracted when the GTC and DTT concentrations were 2.0 M and 80 mM, respectively, 20 μl magnetic beads were added, the incubation temperature was 80 °C, the pH was 8 or 9, and RNA carrier A was used. Therefore, we established a simple method to extract nucleic acids from two important respiratory viruses compared with other commercial kits. This magnetic beads-based co-extraction method for sputum followed by a multiplex RT-qPCR can rapidly and precisely detect DNA and RNA viruses from a single clinical specimen and has many advantages, such as decreased time, low cost, and a lack of harmful chemicals. PMID:28332631

  9. Automation of lidar-based hydrologic feature extraction workflows using GIS

    NASA Astrophysics Data System (ADS)

    Borlongan, Noel Jerome B.; de la Cruz, Roel M.; Olfindo, Nestor T.; Perez, Anjillyn Mae C.

    2016-10-01

    With the advent of LiDAR technology, higher resolution datasets become available for use in different remote sensing and GIS applications. One significant application of LiDAR datasets in the Philippines is in resource features extraction. Feature extraction using LiDAR datasets require complex and repetitive workflows which can take a lot of time for researchers through manual execution and supervision. The Development of the Philippine Hydrologic Dataset for Watersheds from LiDAR Surveys (PHD), a project under the Nationwide Detailed Resources Assessment Using LiDAR (Phil-LiDAR 2) program, created a set of scripts, the PHD Toolkit, to automate its processes and workflows necessary for hydrologic features extraction specifically Streams and Drainages, Irrigation Network, and Inland Wetlands, using LiDAR Datasets. These scripts are created in Python and can be added in the ArcGIS® environment as a toolbox. The toolkit is currently being used as an aid for the researchers in hydrologic feature extraction by simplifying the workflows, eliminating human errors when providing the inputs, and providing quick and easy-to-use tools for repetitive tasks. This paper discusses the actual implementation of different workflows developed by Phil-LiDAR 2 Project 4 in Streams, Irrigation Network and Inland Wetlands extraction.

  10. RNA Preservation Agents and Nucleic Acid Extraction Method Bias Perceived Bacterial Community Composition

    PubMed Central

    McCarthy, Ann; Chiang, Edna; Schmidt, Marian L.; Denef, Vincent J.

    2015-01-01

    Bias is a pervasive problem when characterizing microbial communities. An important source is the difference in lysis efficiencies of different populations, which vary depending on the extraction protocol used. To avoid such biases impacting comparisons between gene and transcript abundances in the environment, the use of one protocol that simultaneously extracts both types of nucleic acids from microbial community samples has gained popularity. However, knowledge regarding tradeoffs to combined nucleic acid extraction protocols is limited, particularly regarding yield and biases in the observed community composition. Here, we evaluated a commercially available protocol for simultaneous extraction of DNA and RNA, which we adapted for freshwater microbial community samples that were collected on filters. DNA and RNA yields were comparable to other commonly used, but independent DNA and RNA extraction protocols. RNA protection agents benefited RNA quality, but decreased DNA yields significantly. Choice of extraction protocol influenced the perceived bacterial community composition, with strong method-dependent biases observed for specific phyla such as the Verrucomicrobia. The combined DNA/RNA extraction protocol detected significantly higher levels of Verrucomicrobia than the other protocols, and those higher numbers were confirmed by microscopic analysis. Use of RNA protection agents as well as independent sequencing runs caused a significant shift in community composition as well, albeit smaller than the shift caused by using different extraction protocols. Despite methodological biases, sample origin was the strongest determinant of community composition. However, when the abundance of specific phylogenetic groups is of interest, researchers need to be aware of the biases their methods introduce. This is particularly relevant if different methods are used for DNA and RNA extraction, in addition to using RNA protection agents only for RNA samples. PMID:25798612

  11. RNA preservation agents and nucleic acid extraction method bias perceived bacterial community composition.

    PubMed

    McCarthy, Ann; Chiang, Edna; Schmidt, Marian L; Denef, Vincent J

    2015-01-01

    Bias is a pervasive problem when characterizing microbial communities. An important source is the difference in lysis efficiencies of different populations, which vary depending on the extraction protocol used. To avoid such biases impacting comparisons between gene and transcript abundances in the environment, the use of one protocol that simultaneously extracts both types of nucleic acids from microbial community samples has gained popularity. However, knowledge regarding tradeoffs to combined nucleic acid extraction protocols is limited, particularly regarding yield and biases in the observed community composition. Here, we evaluated a commercially available protocol for simultaneous extraction of DNA and RNA, which we adapted for freshwater microbial community samples that were collected on filters. DNA and RNA yields were comparable to other commonly used, but independent DNA and RNA extraction protocols. RNA protection agents benefited RNA quality, but decreased DNA yields significantly. Choice of extraction protocol influenced the perceived bacterial community composition, with strong method-dependent biases observed for specific phyla such as the Verrucomicrobia. The combined DNA/RNA extraction protocol detected significantly higher levels of Verrucomicrobia than the other protocols, and those higher numbers were confirmed by microscopic analysis. Use of RNA protection agents as well as independent sequencing runs caused a significant shift in community composition as well, albeit smaller than the shift caused by using different extraction protocols. Despite methodological biases, sample origin was the strongest determinant of community composition. However, when the abundance of specific phylogenetic groups is of interest, researchers need to be aware of the biases their methods introduce. This is particularly relevant if different methods are used for DNA and RNA extraction, in addition to using RNA protection agents only for RNA samples.

  12. Comparative evaluation of total RNA extraction methods in Theobroma cacao using shoot apical meristems.

    PubMed

    Silva, D V; Branco, S M J; Holanda, I S A; Royaert, S; Motamayor, J C; Marelli, J P; Corrêa, R X

    2016-03-04

    Theobroma cacao is a species of great economic importance with its beans used for chocolate production. The tree has been a target of various molecular studies. It contains many polyphenols, which complicate the extraction of nucleic acids with the extraction protocols requiring a large amount of plant material. These issues, therefore, necessitate the optimization of the protocols. The aim of the present study was to evaluate different methods for extraction of total RNA from shoot apical meristems of T. cacao 'CCN 51' and to assess the influence of storage conditions for the meristems on the extraction. The study also aimed to identify the most efficient protocol for RNA extraction using a small amount of plant material. Four different protocols were evaluated for RNA extraction using one shoot apical meristem per sample. Among these protocols, one that was more efficient was then tested to extract RNA using four different numbers of shoot apical meristems, subjected to three different storage conditions. The best protocol was tested for cDNA amplification using reverse transcription-polymerase chain reaction; the cDNA quality was determined to be satisfactory for molecular analyses. The study revealed that with the best RNA extraction protocol, one shoot apical meristem was sufficient for extraction of high-quality total RNA. The results obtained might enable advances in genetic analyses and molecular studies using reduced amount of plant material.

  13. Strategies for Medical Data Extraction and Presentation Part 3: Automated Context- and User-Specific Data Extraction.

    PubMed

    Reiner, Bruce

    2015-08-01

    In current medical practice, data extraction is limited by a number of factors including lack of information system integration, manual workflow, excessive workloads, and lack of standardized databases. The combined limitations result in clinically important data often being overlooked, which can adversely affect clinical outcomes through the introduction of medical error, diminished diagnostic confidence, excessive utilization of medical services, and delays in diagnosis and treatment planning. Current technology development is largely inflexible and static in nature, which adversely affects functionality and usage among the diverse and heterogeneous population of end users. In order to address existing limitations in medical data extraction, alternative technology development strategies need to be considered which incorporate the creation of end user profile groups (to account for occupational differences among end users), customization options (accounting for individual end user needs and preferences), and context specificity of data (taking into account both the task being performed and data subject matter). Creation of the proposed context- and user-specific data extraction and presentation templates offers a number of theoretical benefits including automation and improved workflow, completeness in data search, ability to track and verify data sources, creation of computerized decision support and learning tools, and establishment of data-driven best practice guidelines.

  14. CHANNEL MORPHOLOGY TOOL (CMT): A GIS-BASED AUTOMATED EXTRACTION MODEL FOR CHANNEL GEOMETRY

    SciTech Connect

    JUDI, DAVID; KALYANAPU, ALFRED; MCPHERSON, TIMOTHY; BERSCHEID, ALAN

    2007-01-17

    This paper describes an automated Channel Morphology Tool (CMT) developed in ArcGIS 9.1 environment. The CMT creates cross-sections along a stream centerline and uses a digital elevation model (DEM) to create station points with elevations along each of the cross-sections. The generated cross-sections may then be exported into a hydraulic model. Along with the rapid cross-section generation the CMT also eliminates any cross-section overlaps that might occur due to the sinuosity of the channels using the Cross-section Overlap Correction Algorithm (COCoA). The CMT was tested by extracting cross-sections from a 5-m DEM for a 50-km channel length in Houston, Texas. The extracted cross-sections were compared directly with surveyed cross-sections in terms of the cross-section area. Results indicated that the CMT-generated cross-sections satisfactorily matched the surveyed data.

  15. Automated extraction of subdural electrode grid from post-implant MRI scans for epilepsy surgery

    NASA Astrophysics Data System (ADS)

    Pozdin, Maksym A.; Skrinjar, Oskar

    2005-04-01

    This paper presents an automated algorithm for extraction of Subdural Electrode Grid (SEG) from post-implant MRI scans for epilepsy surgery. Post-implant MRI scans are corrupted by the image artifacts caused by implanted electrodes. The artifacts appear as dark spherical voids and given that the cerebrospinal fluid is also dark in T1-weigthed MRI scans, it is a difficult and time-consuming task to manually locate SEG position relative to brain structures of interest. The proposed algorithm reliably and accurately extracts SEG from post-implant MRI scan, i.e. finds its shape and position relative to brain structures of interest. The algorithm was validated against manually determined electrode locations, and the average error was 1.6mm for the three tested subjects.

  16. Modelling and representation issues in automated feature extraction from aerial and satellite images

    NASA Astrophysics Data System (ADS)

    Sowmya, Arcot; Trinder, John

    New digital systems for the processing of photogrammetric and remote sensing images have led to new approaches to information extraction for mapping and Geographic Information System (GIS) applications, with the expectation that data can become more readily available at a lower cost and with greater currency. Demands for mapping and GIS data are increasing as well for environmental assessment and monitoring. Hence, researchers from the fields of photogrammetry and remote sensing, as well as computer vision and artificial intelligence, are bringing together their particular skills for automating these tasks of information extraction. The paper will review some of the approaches used in knowledge representation and modelling for machine vision, and give examples of their applications in research for image understanding of aerial and satellite imagery.

  17. Image-based continental shelf habitat mapping using novel automated data extraction techniques

    NASA Astrophysics Data System (ADS)

    Seiler, Jan; Friedman, Ariell; Steinberg, Daniel; Barrett, Neville; Williams, Alan; Holbrook, Neil J.

    2012-08-01

    We automatically mapped the distribution of temperate continental shelf rocky reef habitats with a high degree of confidence using colour, texture, rugosity and patchiness features extracted from images in conjunction with machine-learning algorithms. This demonstrated the potential of novel automation routines to expedite the complex and time-consuming process of seabed mapping. The random forests ensemble classifier outperformed other tree-based algorithms and also offered some valuable built-in model performance assessment tools. Habitat prediction using random forests performed most accurately when all 26 image-derived predictors were included in the model. This produced an overall habitat prediction accuracy of 84% (with a kappa statistic of 0.793) when compared to nine distinct habitat classes assigned by a human annotator. Predictions for three habitat classes were all within the 95% confidence intervals, indicating close agreement between observed and predicted habitat classes. Misclassified images were mostly unevenly, partially or insufficiently illuminated and came mostly from rugged terrains and during the autonomous underwater vehicle's obstacle avoidance manoeuvres. The remaining misclassified images were wrongly or inconsistently labelled by the human annotator. This study demonstrates the suitability of autonomous underwater vehicles to effectively sample benthic habitats and the ability of automated data handling techniques to extract and reliably process large volumes of seabed image data. Our methods for image feature extraction and classification are repeatable, cost-effective and well suited to studies that require non-extractive and/or co-located sampling, e.g. in marine reserves and for monitoring the recovery from physical impacts, e.g. from bottom fishing activities. The methods are transferable to other continental shelf areas and to other disciplines such as seabed geology.

  18. OMIT: Dynamic, Semi-Automated Ontology Development for the microRNA Domain

    PubMed Central

    Huang, Jingshan; Dang, Jiangbo; Borchert, Glen M.; Eilbeck, Karen; Zhang, He; Xiong, Min; Jiang, Weijian; Wu, Hao; Blake, Judith A.; Natale, Darren A.; Tan, Ming

    2014-01-01

    As a special class of short non-coding RNAs, microRNAs (a.k.a. miRNAs or miRs) have been reported to perform important roles in various biological processes by regulating respective target genes. However, significant barriers exist during biologists' conventional miR knowledge discovery. Emerging semantic technologies, which are based upon domain ontologies, can render critical assistance to this problem. Our previous research has investigated the construction of a miR ontology, named Ontology for MIcroRNA Target Prediction (OMIT), the very first of its kind that formally encodes miR domain knowledge. Although it is unavoidable to have a manual component contributed by domain experts when building ontologies, many challenges have been identified for a completely manual development process. The most significant issue is that a manual development process is very labor-intensive and thus extremely expensive. Therefore, we propose in this paper an innovative ontology development methodology. Our contributions can be summarized as: (i) We have continued the development and critical improvement of OMIT, solidly based on our previous research outcomes. (ii) We have explored effective and efficient algorithms with which the ontology development can be seamlessly combined with machine intelligence and be accomplished in a semi-automated manner, thus significantly reducing large amounts of human efforts. A set of experiments have been conducted to thoroughly evaluate our proposed methodology. PMID:25025130

  19. OMIT: dynamic, semi-automated ontology development for the microRNA domain.

    PubMed

    Huang, Jingshan; Dang, Jiangbo; Borchert, Glen M; Eilbeck, Karen; Zhang, He; Xiong, Min; Jiang, Weijian; Wu, Hao; Blake, Judith A; Natale, Darren A; Tan, Ming

    2014-01-01

    As a special class of short non-coding RNAs, microRNAs (a.k.a. miRNAs or miRs) have been reported to perform important roles in various biological processes by regulating respective target genes. However, significant barriers exist during biologists' conventional miR knowledge discovery. Emerging semantic technologies, which are based upon domain ontologies, can render critical assistance to this problem. Our previous research has investigated the construction of a miR ontology, named Ontology for MIcroRNA Target Prediction (OMIT), the very first of its kind that formally encodes miR domain knowledge. Although it is unavoidable to have a manual component contributed by domain experts when building ontologies, many challenges have been identified for a completely manual development process. The most significant issue is that a manual development process is very labor-intensive and thus extremely expensive. Therefore, we propose in this paper an innovative ontology development methodology. Our contributions can be summarized as: (i) We have continued the development and critical improvement of OMIT, solidly based on our previous research outcomes. (ii) We have explored effective and efficient algorithms with which the ontology development can be seamlessly combined with machine intelligence and be accomplished in a semi-automated manner, thus significantly reducing large amounts of human efforts. A set of experiments have been conducted to thoroughly evaluate our proposed methodology.

  20. Automated solid-phase extraction approaches for large scale biomonitoring studies.

    PubMed

    Kuklenyik, Zsuzsanna; Ye, Xiaoyun; Needham, Larry L; Calafat, Antonia M

    2009-01-01

    The main value in measuring environmental chemicals in biological specimens (i.e., biomonitoring) is the ability to minimize risk assessment uncertainties. The collection of biomonitoring data for risk assessment requires the analysis of a statistically significant number of samples from subjects with a significant prevalence of detectable internal dose levels. This paper addresses the practical laboratory challenges that arise from these statistical requirements: development of high throughput techniques that can handle, with high accuracy and precision, a large number of samples and can do a trace level analysis of multiple and diverse environmental chemicals (i.e., analytes). We review here examples of high throughput, automated solid-phase extraction methods developed in our laboratory for biomonitoring of analytes with representative hydrophobic properties and for typical biomonitoring matrices. We discuss key aspects of sample preparation, column, and solvent selection for off- and online extractions, and the so-called nuts-and-bolts of online column-switching systems necessary for developing-with minimal sample handling-rugged, automated methods.

  1. A non-phenol-chloroform extraction of double-stranded RNA from plant and fungal tissues.

    PubMed

    Balijja, Alitukiriza; Kvarnheden, Anders; Turchetti, Tullio

    2008-09-01

    Double-stranded RNA (dsRNA) molecules of viruses are found in nature at a very high frequency. Their detection in plants and fungi has been carried out with difficulty due to the complicated dsRNA extraction techniques used commonly which includes phenol-chloroform extractions. In this study, an extraction method for isolation of dsRNA is described that is free of phenol and chloroform. A lysis buffer, containing beta-mercaptoethanol and polyvinylpolypyrrolidone (PVPP-40), was added to homogenised tissues and the subsequent supernatant was filtered through a cellulose CF-11 mini-column. DsRNA molecules were separated based on the differing affinity of nucleic acids for the cellulose CF-11 resin in 20% ethanol buffer. This easy, rapid and cheap technique has been successfully tested on fungi and plants containing different dsRNA virus molecules, indicating the possibility of a wide use of the method.

  2. Simultaneous Extraction from Bacterioplankton of Total RNA and DNA Suitable for Quantitative Structure and Function Analyses

    PubMed Central

    Weinbauer, Markus G.; Fritz, Ingo; Wenderoth, Dirk F.; Höfle, Manfred G.

    2002-01-01

    The aim of this study was to develop a protocol for the simultaneous extraction from bacterioplankton of RNA and DNA suitable for quantitative molecular analysis. By using a combined mechanical and chemical extraction method, the highest RNA and DNA yield was obtained with sodium lauryl sarcosinate-phenol or DivoLab-phenol as the extraction mix. The efficiency of extraction of nucleic acids was comparatively high and varied only moderately in gram-negative bacterial isolates and bacterioplankton (RNA, 52 to 66%; DNA, 43 to 61%); significant amounts of nucleic acids were also obtained for a gram-positive bacterial isolate (RNA, 20 to 30%; DNA, 20 to 25%). Reverse transcription-PCR and PCR amplification products of fragments of 16S rRNA and its genes were obtained from all isolates and communities, indicating that the extracted nucleic acids were intact and pure enough for community structure analyses. By using single-strand conformation polymorphism of fragments of 16S rRNA and its gene, community fingerprints were obtained from pond bacterioplankton. mRNA transcripts encoding fragments of the enzyme nitrite reductase gene (nir gene) could be detected in a pond water sample, indicating that the extraction method is also suitable for studying gene expression. The extraction method presented yields nucleic acids that can be used to perform structural and functional studies of bacterioplankton communities from a single sample. PMID:11872453

  3. Automated sample preparation by pressurized liquid extraction-solid-phase extraction for the liquid chromatographic-mass spectrometric investigation of polyphenols in the brewing process.

    PubMed

    Papagiannopoulos, Menelaos; Mellenthin, Annett

    2002-11-08

    The analysis of polyphenols from solid plant or food samples usually requires laborious sample preparation. The liquid extraction of these compounds from the sample is compromised by apolar matrix interferences, an excess of which has to be eliminated prior to subsequent purification and separation. Applying pressurized liquid extraction to the extraction of polyphenols from hops, the use of different solvents sequentially can partly overcome these problems. Initial extraction with pentane eliminates hydrophobic compounds like hop resins and oils and enables the straightforward automated on-line solid-phase extraction as part of an optimized LC-MS analysis.

  4. ChemDataExtractor: A Toolkit for Automated Extraction of Chemical Information from the Scientific Literature.

    PubMed

    Swain, Matthew C; Cole, Jacqueline M

    2016-10-24

    The emergence of "big data" initiatives has led to the need for tools that can automatically extract valuable chemical information from large volumes of unstructured data, such as the scientific literature. Since chemical information can be present in figures, tables, and textual paragraphs, successful information extraction often depends on the ability to interpret all of these domains simultaneously. We present a complete toolkit for the automated extraction of chemical entities and their associated properties, measurements, and relationships from scientific documents that can be used to populate structured chemical databases. Our system provides an extensible, chemistry-aware, natural language processing pipeline for tokenization, part-of-speech tagging, named entity recognition, and phrase parsing. Within this scope, we report improved performance for chemical named entity recognition through the use of unsupervised word clustering based on a massive corpus of chemistry articles. For phrase parsing and information extraction, we present the novel use of multiple rule-based grammars that are tailored for interpreting specific document domains such as textual paragraphs, captions, and tables. We also describe document-level processing to resolve data interdependencies and show that this is particularly necessary for the autogeneration of chemical databases since captions and tables commonly contain chemical identifiers and references that are defined elsewhere in the text. The performance of the toolkit to correctly extract various types of data was evaluated, affording an F-score of 93.4%, 86.8%, and 91.5% for extracting chemical identifiers, spectroscopic attributes, and chemical property attributes, respectively; set against the CHEMDNER chemical name extraction challenge, ChemDataExtractor yields a competitive F-score of 87.8%. All tools have been released under the MIT license and are available to download from http://www.chemdataextractor.org .

  5. An automated RNA-Seq analysis pipeline to identify and visualize differentially expressed genes and pathways in CHO cells.

    PubMed

    Chen, Chun; Le, Huong; Goudar, Chetan T

    2015-01-01

    Recent advances in RNA-Seq based comparative transcriptomics have opened up a unique opportunity to understand the mechanisms of different phenotypes in bioprocessing-related cell lines including Chinese hamster ovary (CHO) cells. However, simple and powerful tools are needed to translate large data sets into biologically relevant information that can be leveraged for genetic engineering and cell culture medium and process development. While tools exist to perform specific tasks associated with transcriptomics analysis, integrated end to end solutions that span the entire spectrum of raw data processing to visualization of gene expression changes on canonical pathways are rare. Additionally, these are not automated and require substantial user intervention. To address this gap, we have developed an automated RNA-Seq analysis pipeline in R which leverages the latest public domain statistical advances in transcriptomics data analysis. This pipeline reads RNA-Seq gene count data, identifies differentially expressed genes and differentially expressed pathways, and provides multiple intuitive visualizations as outputs. By using two publicly available CHO RNA-Seq datasets, we have demonstrated the utility of this pipeline. Subsequently, this pipeline was used to demonstrate transcriptomic similarity between laboratory- and pilot-scale bioreactors, helping make a case for the suitability of the lab-scale bioreactor as a scaled-down model. Automated end to end RNA-Seq data analysis approaches such as the one presented in this study will shorten the time required from acquiring sequencing data to biological interpretation of the results and can help accelerate the adoption of RNA-Seq analysis and thus mechanism-driven approaches for cell line and bioprocess optimization. © 2015 American Institute of Chemical Engineers.

  6. High-quality RNA extraction from copepods for Next Generation Sequencing: A comparative study.

    PubMed

    Asai, Sneha; Ianora, Adrianna; Lauritano, Chiara; Lindeque, Penelope K; Carotenuto, Ylenia

    2015-12-01

    Despite the ecological importance of copepods, few Next Generation Sequencing studies (NGS) have been performed on small crustaceans, and a standard method for RNA extraction is lacking. In this study, we compared three commonly-used methods: TRIzol®, Aurum Total RNA Mini Kit and Qiagen RNeasy Micro Kit, in combination with preservation reagents TRIzol® or RNAlater®, to obtain high-quality and quantity of RNA from copepods for NGS. Total RNA was extracted from the copepods Calanus helgolandicus, Centropages typicus and Temora stylifera and its quantity and quality were evaluated using NanoDrop, agarose gel electrophoresis and Agilent Bioanalyzer. Our results demonstrate that preservation of copepods in RNAlater® and extraction with Qiagen RNeasy Micro Kit were the optimal isolation method for high-quality and quantity of RNA for NGS studies of C. helgolandicus. Intriguingly, C. helgolandicus 28S rRNA is formed by two subunits that separate after heat-denaturation and migrate along with 18S rRNA. This unique property of protostome RNA has never been reported in copepods. Overall, our comparative study on RNA extraction protocols will help increase gene expression studies on copepods using high-throughput applications, such as RNA-Seq and microarrays. Copyright © 2014 Elsevier B.V. All rights reserved.

  7. Extraction and comparison of gene expression patterns from 2D RNA in situ hybridization images.

    PubMed

    Mace, Daniel L; Varnado, Nicole; Zhang, Weiping; Frise, Erwin; Ohler, Uwe

    2010-03-15

    Recent advancements in high-throughput imaging have created new large datasets with tens of thousands of gene expression images. Methods for capturing these spatial and/or temporal expression patterns include in situ hybridization or fluorescent reporter constructs or tags, and results are still frequently assessed by subjective qualitative comparisons. In order to deal with available large datasets, fully automated analysis methods must be developed to properly normalize and model spatial expression patterns. We have developed image segmentation and registration methods to identify and extract spatial gene expression patterns from RNA in situ hybridization experiments of Drosophila embryos. These methods allow us to normalize and extract expression information for 78,621 images from 3724 genes across six time stages. The similarity between gene expression patterns is computed using four scoring metrics: mean squared error, Haar wavelet distance, mutual information and spatial mutual information (SMI). We additionally propose a strategy to calculate the significance of the similarity between two expression images, by generating surrogate datasets with similar spatial expression patterns using a Monte Carlo swap sampler. On data from an early development time stage, we show that SMI provides the most biologically relevant metric of comparison, and that our significance testing generalizes metrics to achieve similar performance. We exemplify the application of spatial metrics on the well-known Drosophila segmentation network. A Java webstart application to register and compare patterns, as well as all source code, are available from: http://tools.genome.duke.edu/generegulation/image_analysis/insitu uwe.ohler@duke.edu Supplementary data are available at Bioinformatics online.

  8. Automated hand thermal image segmentation and feature extraction in the evaluation of rheumatoid arthritis.

    PubMed

    Snekhalatha, U; Anburajan, M; Sowmiya, V; Venkatraman, B; Menaka, M

    2015-04-01

    The aim of the study was (1) to perform an automated segmentation of hot spot regions of the hand from thermograph using the k-means algorithm and (2) to test the potential of features extracted from the hand thermograph and its measured skin temperature indices in the evaluation of rheumatoid arthritis. Thermal image analysis based on skin temperature measurement, heat distribution index and thermographic index was analyzed in rheumatoid arthritis patients and controls. The k-means algorithm was used for image segmentation, and features were extracted from the segmented output image using the gray-level co-occurrence matrix method. In metacarpo-phalangeal, proximal inter-phalangeal and distal inter-phalangeal regions, the calculated percentage difference in the mean values of skin temperatures was found to be higher in rheumatoid arthritis patients (5.3%, 4.9% and 4.8% in MCP3, PIP3 and DIP3 joints, respectively) as compared to the normal group. k-Means algorithm applied in the thermal imaging provided better segmentation results in evaluating the disease. In the total population studied, the measured mean average skin temperature of the MCP3 joint was highly correlated with most of the extracted features of the hand. In the total population studied, the statistical feature extracted parameters correlated significantly with skin surface temperature measurements and measured temperature indices. Hence, the developed computer-aided diagnostic tool using MATLAB could be used as a reliable method in diagnosing and analyzing the arthritis in hand thermal images.

  9. Automated Detection and Extraction of Coronal Dimmings from SDO/AIA Data

    NASA Astrophysics Data System (ADS)

    Davey, Alisdair R.; Attrill, G. D. R.; Wills-Davey, M. J.

    2010-05-01

    The sheer volume of data anticipated from the Solar Dynamics Observatory/Atmospheric Imaging Assembly (SDO/AIA) highlights the necessity for the development of automatic detection methods for various types of solar activity. Initially recognised in the 1970s, it is now well established that coronal dimmings are closely associated with coronal mass ejections (CMEs), and are particularly recognised as an indicator of front-side (halo) CMEs, which can be difficult to detect in white-light coronagraph data. An automated coronal dimming region detection and extraction algorithm removes visual observer bias from determination of physical quantities such as spatial location, area and volume. This allows reproducible, quantifiable results to be mined from very large datasets. The information derived may facilitate more reliable early space weather detection, as well as offering the potential for conducting large-sample studies focused on determining the geoeffectiveness of CMEs, coupled with analysis of their associated coronal dimmings. We present examples of dimming events extracted using our algorithm from existing EUV data, demonstrating the potential for the anticipated application to SDO/AIA data. Metadata returned by our algorithm include: location, area, volume, mass and dynamics of coronal dimmings. As well as running on historic datasets, this algorithm is capable of detecting and extracting coronal dimmings in near real-time. The coronal dimming detection and extraction algorithm described in this poster is part of the SDO/Computer Vision Center effort hosted at SAO (Martens et al., 2009). We acknowledge NASA grant NNH07AB97C.

  10. Hybrid curation of gene–mutation relations combining automated extraction and crowdsourcing

    PubMed Central

    Burger, John D.; Doughty, Emily; Khare, Ritu; Wei, Chih-Hsuan; Mishra, Rajashree; Aberdeen, John; Tresner-Kirsch, David; Wellner, Ben; Kann, Maricel G.; Lu, Zhiyong; Hirschman, Lynette

    2014-01-01

    Background: This article describes capture of biological information using a hybrid approach that combines natural language processing to extract biological entities and crowdsourcing with annotators recruited via Amazon Mechanical Turk to judge correctness of candidate biological relations. These techniques were applied to extract gene– mutation relations from biomedical abstracts with the goal of supporting production scale capture of gene–mutation–disease findings as an open source resource for personalized medicine. Results: The hybrid system could be configured to provide good performance for gene–mutation extraction (precision ∼82%; recall ∼70% against an expert-generated gold standard) at a cost of $0.76 per abstract. This demonstrates that crowd labor platforms such as Amazon Mechanical Turk can be used to recruit quality annotators, even in an application requiring subject matter expertise; aggregated Turker judgments for gene–mutation relations exceeded 90% accuracy. Over half of the precision errors were due to mismatches against the gold standard hidden from annotator view (e.g. incorrect EntrezGene identifier or incorrect mutation position extracted), or incomplete task instructions (e.g. the need to exclude nonhuman mutations). Conclusions: The hybrid curation model provides a readily scalable cost-effective approach to curation, particularly if coupled with expert human review to filter precision errors. We plan to generalize the framework and make it available as open source software. Database URL: http://www.mitre.org/publications/technical-papers/hybrid-curation-of-gene-mutation-relations-combining-automated PMID:25246425

  11. An automated system for liquid-liquid extraction in monosegmented flow analysis

    PubMed Central

    Facchin, Ileana; Pasquini, Celio

    1997-01-01

    An automated system to perform liquid-liquid extraction in monosegmented flow analysis is described. The system is controlled by a microcomputer that can track the localization of the aqueous monosegmented sample in the manifold. Optical switches are employed to sense the gas-liquid interface of the air bubbles that define the monosegment. The logical level changes, generated by the switches, are flagged by the computer through a home-made interface that also contains the analogue-to-digital converter for signal acquisition. The sequence of operations, necessary for a single extraction or for concentration of the analyte in the organic phase, is triggered by these logical transitions. The system was evaluated for extraction of Cd(II), Cu(II) and Zn(II) and concentration of Cd(II) from aqueous solutions at pH 9.9 (NH3/NH4Cl buffer) into chloroform containing PAN (1-(2-pyridylazo)-2-naphthol) . The results show a mean repeatability of 3% (rsd) for a 2.0 mg l-1 Cd(II) solution and a linear increase of the concentration factor for a 0.5mg l-1 Cd(II) solution observed for up to nine extraction cycles. PMID:18924792

  12. Evidence for an RNA Polymerization Activity in Axolotl and Xenopus Egg Extracts

    PubMed Central

    Pelczar, Hélène; Woisard, Anne; Lemaître, Jean Marc; Chachou, Mohamed; Andéol, Yannick

    2010-01-01

    We have previously reported a post-transcriptional RNA amplification observed in vivo following injection of in vitro synthesized transcripts into axolotl oocytes, unfertilized (UFE) or fertilized eggs. To further characterize this phenomenon, low speed extracts (LSE) from axolotl and Xenopus UFE were prepared and tested in an RNA polymerization assay. The major conclusions are: i) the amphibian extracts catalyze the incorporation of radioactive ribonucleotide in RNase but not DNase sensitive products showing that these products correspond to RNA; ii) the phenomenon is resistant to α-amanitin, an inhibitor of RNA polymerases II and III and to cordycepin (3′dAMP), but sensitive to cordycepin 5′-triphosphate, an RNA elongation inhibitor, which supports the existence of an RNA polymerase activity different from polymerases II and III; the detection of radiolabelled RNA comigrating at the same length as the exogenous transcript added to the extracts allowed us to show that iii) the RNA polymerization is not a 3′ end labelling and that iv) the radiolabelled RNA is single rather than double stranded. In vitro cell-free systems derived from amphibian UFE therefore validate our previous in vivo results hypothesizing the existence of an evolutionary conserved enzymatic activity with the properties of an RNA dependent RNA polymerase (RdRp). PMID:21203452

  13. Direct Detection of 16S rRNA in Soil Extracts by Using Oligonucleotide Microarrays

    PubMed Central

    Small, Jack; Call, Douglas R.; Brockman, Fred J.; Straub, Timothy M.; Chandler, Darrell P.

    2001-01-01

    We report on the development and validation of a simple microarray method for the direct detection of intact 16S rRNA from unpurified soil extracts. Total RNAs from Geobacter chapellei and Desulfovibrio desulfuricans were hybridized to an oligonucleotide array consisting of universal and species-specific 16S rRNA probes. PCR-amplified products from Geobacter and Desulfovibrio were easily and specifically detected under a range of hybridization times, temperatures, and buffers. However, reproducible, specific hybridization and detection of intact rRNA could be accomplished only by using a chaperone-detector probe strategy. With this knowledge, assay conditions were developed for rRNA detection using a 2-h hybridization time at room temperature. Hybridization specificity and signal intensity were enhanced using fragmented RNA. Formamide was required in the hybridization buffer in order to achieve species-specific detection of intact rRNA. With the chaperone detection strategy, we were able to specifically hybridize and detect G. chapellei 16S rRNA directly from a total-RNA soil extract, without further purification or removal of soluble soil constituents. The detection sensitivity for G. chapellei 16S rRNA in soil extracts was at least 0.5 μg of total RNA, representing approximately 7.5 × 106 Geobacter cell equivalents of RNA. These results suggest that it is now possible to apply microarray technology to the direct detection of microorganisms in environmental samples, without using PCR. PMID:11571176

  14. A Novel Validation Algorithm Allows for Automated Cell Tracking and the Extraction of Biologically Meaningful Parameters

    PubMed Central

    Madany Mamlouk, Amir; Schicktanz, Simone; Kruse, Charli

    2011-01-01

    Automated microscopy is currently the only method to non-invasively and label-free observe complex multi-cellular processes, such as cell migration, cell cycle, and cell differentiation. Extracting biological information from a time-series of micrographs requires each cell to be recognized and followed through sequential microscopic snapshots. Although recent attempts to automatize this process resulted in ever improving cell detection rates, manual identification of identical cells is still the most reliable technique. However, its tedious and subjective nature prevented tracking from becoming a standardized tool for the investigation of cell cultures. Here, we present a novel method to accomplish automated cell tracking with a reliability comparable to manual tracking. Previously, automated cell tracking could not rival the reliability of manual tracking because, in contrast to the human way of solving this task, none of the algorithms had an independent quality control mechanism; they missed validation. Thus, instead of trying to improve the cell detection or tracking rates, we proceeded from the idea to automatically inspect the tracking results and accept only those of high trustworthiness, while rejecting all other results. This validation algorithm works independently of the quality of cell detection and tracking through a systematic search for tracking errors. It is based only on very general assumptions about the spatiotemporal contiguity of cell paths. While traditional tracking often aims to yield genealogic information about single cells, the natural outcome of a validated cell tracking algorithm turns out to be a set of complete, but often unconnected cell paths, i.e. records of cells from mitosis to mitosis. This is a consequence of the fact that the validation algorithm takes complete paths as the unit of rejection/acceptance. The resulting set of complete paths can be used to automatically extract important biological parameters with high

  15. An efficient RNA extraction method for estimating gut microbial diversity by polymerase chain reaction.

    PubMed

    Kang, Seungha; Denman, Stuart E; Morrison, Mark; Yu, Zhongtang; McSweeney, Chris S

    2009-05-01

    An extraction method was developed to recover high-quality RNA from rumen digesta and mouse feces for phylogenetic analysis of metabolically active members of the gut microbial community. Four extraction methods were tested on different amounts of the same samples and compared for efficiency of recovery and purity of RNA. Trizol extraction after bead beating produced a higher quantity and quality of RNA than a similar method using phenol/chloroform. Dissociation solution produced a 1.5- to 2-fold increase in RNA recovery compared with phosphate-buffered saline during the dissociation of microorganisms from rumen digesta or fecal particles. The identity of metabolically active bacteria in the samples was analyzed by sequencing 87 amplicons produced using bacteria-specific 16S rDNA primers, with cDNA synthesized from the extracted RNA as the template. Amplicons representing the major phyla encountered in the rumen (Firmicutes, 43.7%; Proteobacteria, 28.7%; Bacteroidetes, 25.3%; Spirochea, 1.1%, and Synergistes, 1.1%) were recovered, showing that development of the RNA extraction method enables RNA-based analysis of metabolically active bacterial groups from the rumen and other environments. Interestingly, in rumen samples, about 30% of the sequenced random 16S rRNA amplicons were related to the Proteobacteria, providing the first evidence that this group may have greater importance in rumen metabolism than previously attributed by DNA-based analysis.

  16. Identification of extracellular miRNA in archived serum samples by next-generation sequencing from RNA extracted using multiple methods.

    PubMed

    Gautam, Aarti; Kumar, Raina; Dimitrov, George; Hoke, Allison; Hammamieh, Rasha; Jett, Marti

    2016-10-01

    miRNAs act as important regulators of gene expression by promoting mRNA degradation or by attenuating protein translation. Since miRNAs are stably expressed in bodily fluids, there is growing interest in profiling these miRNAs, as it is minimally invasive and cost-effective as a diagnostic matrix. A technical hurdle in studying miRNA dynamics is the ability to reliably extract miRNA as small sample volumes and low RNA abundance create challenges for extraction and downstream applications. The purpose of this study was to develop a pipeline for the recovery of miRNA using small volumes of archived serum samples. The RNA was extracted employing several widely utilized RNA isolation kits/methods with and without addition of a carrier. The small RNA library preparation was carried out using Illumina TruSeq small RNA kit and sequencing was carried out using Illumina platform. A fraction of five microliters of total RNA was used for library preparation as quantification is below the detection limit. We were able to profile miRNA levels in serum from all the methods tested. We found out that addition of nucleic acid based carrier molecules had higher numbers of processed reads but it did not enhance the mapping of any miRBase annotated sequences. However, some of the extraction procedures offer certain advantages: RNA extracted by TRIzol seemed to align to the miRBase best; extractions using TRIzol with carrier yielded higher miRNA-to-small RNA ratios. Nuclease free glycogen can be carrier of choice for miRNA sequencing. Our findings illustrate that miRNA extraction and quantification is influenced by the choice of methodologies. Addition of nucleic acid- based carrier molecules during extraction procedure is not a good choice when assaying miRNA using sequencing. The careful selection of an extraction method permits the archived serum samples to become valuable resources for high-throughput applications.

  17. Evaluation of commercial kits for extraction of DNA and RNA from Clostridium difficile.

    PubMed

    Metcalf, Devon; Weese, J Scott

    2012-12-01

    Commercial nucleic acid extraction kits are a cost effective, efficient and convenient way to isolate DNA and RNA from bacteria. Despite the increasing importance of the gastrointestinal pathogen, Clostridium difficile, and the increased use of nucleic acids in its identification, characterization, and investigation of virulence factors, no standardized or recommended methods for nucleic acid isolation exist. Here, we sought to evaluate 4 commercial DNA extraction kits and 3 commercial RNA extraction kits assessing cost, labor intensity, purity, quantity and quality of nucleic acid preparations. The DNA extraction kits produced a range of concentrations (20.9-546 ng/ml) and A(260/280) ratios (1.92-2.11). All kits were suitable for DNA extraction with the exception of the Roche MagNA pure LC DNA isolation kit III which produced DNA of high yield but with substantial shearing, but that did not affect downstream PCR amplifications. For RNA extraction, the Qiagen RNeasy mini kit stood out producing preparations of consistently higher concentrations and higher RNA integrity numbers (RIN). The Roche MagNA pure LC RNA isolation kit produced preparations that could not be properly assigned RINs due to a failure to remove small RNAs which were interpreted as degradation. Good DNA and RNA yield are critical but methods are often overlooked. This study highlights the potential for critical variation between established commercial systems and the need for assessment of any extraction methods that are used.

  18. Rapid and reliable method of extracting DNA and RNA from sweetpotato, Ipomoea batatas (L). Lam.

    PubMed

    Kim, Sun-Hyung; Hamada, Tatsuro

    2005-12-01

    A quick, simple and reliable method of extracting DNA from sweetpotato (Ipomoea batatas (L.) Lam.) has been developed. The method was applied successfully for extraction of total DNA from leaves and total RNA from leaves and various tissues. The yield of DNA extracted by this procedure was high (about 1 mg/g leaf tissue). The extracted DNA was completely digested by restriction endonucleases indicating the absence of common contaminating compounds. The absorbancy ratios of A260/A230 and A260/A280 of isolated RNA were approx. 2 and the yield was about 0.2 mg/g fresh wt. CIPK and tublin genes were successfully amplified by RT-PCR, suggesting the integrity of isolated RNA. The total DNA and RNA isolated by this method was of sufficient quality for subsequent molecular analysis.

  19. Semi-automated extraction of longitudinal subglacial bedforms from digital terrain models - Two new methods

    NASA Astrophysics Data System (ADS)

    Jorge, Marco G.; Brennand, Tracy A.

    2017-07-01

    Relict drumlin and mega-scale glacial lineation (positive relief, longitudinal subglacial bedforms - LSBs) morphometry has been used as a proxy for paleo ice-sheet dynamics. LSB morphometric inventories have relied on manual mapping, which is slow and subjective and thus potentially difficult to reproduce. Automated methods are faster and reproducible, but previous methods for LSB semi-automated mapping have not been highly successful. Here, two new object-based methods for the semi-automated extraction of LSBs (footprints) from digital terrain models are compared in a test area in the Puget Lowland, Washington, USA. As segmentation procedures to create LSB-candidate objects, the normalized closed contour method relies on the contouring of a normalized local relief model addressing LSBs on slopes, and the landform elements mask method relies on the classification of landform elements derived from the digital terrain model. For identifying which LSB-candidate objects correspond to LSBs, both methods use the same LSB operational definition: a ruleset encapsulating expert knowledge, published morphometric data, and the morphometric range of LSBs in the study area. The normalized closed contour method was separately applied to four different local relief models, two computed in moving windows and two hydrology-based. Overall, the normalized closed contour method outperformed the landform elements mask method. The normalized closed contour method performed on a hydrological relief model from a multiple direction flow routing algorithm performed best. For an assessment of its transferability, the normalized closed contour method was evaluated on a second area, the Chautauqua drumlin field, Pennsylvania and New York, USA where it performed better than in the Puget Lowland. A broad comparison to previous methods suggests that the normalized relief closed contour method may be the most capable method to date, but more development is required.

  20. Extraction of RNA from fresh, frozen, and lyophilized tuber and root tissues.

    PubMed

    Kumar, G N Mohan; Iyer, Suresh; Knowles, N Richard

    2007-03-07

    A method for isolating transcriptionally competent RNA from fresh, frozen, and lyophilized plant storage tissues containing high levels of starch and phenolics is described. The protocol avoids the use of guanidium salts, which often lead to the formation of a viscous gel during extraction of high starch-containing tissues, and instead uses a borate-Tris buffer in combination with high concentrations of NaCl, Na2SO3, and sodium dodecyl sulfate in the extraction medium. RNA was extracted from fresh, frozen, and lyophilized tissues of potato tubers, storage roots of sweet potato, radish, and turnip, and rhizomes of ginger. The yield of RNA from potato tubers averaged 281 microg g fresh weight(-1) and 1584 microg g dry weight(-1) from frozen and lyophilized samples, respectively. A260/A230 ratios of potato RNA extracts were 2.2 or greater, indicating minimal contamination by polyphenols and carbohydrates. Similarly, A260/A280 ratios exceeded 1.9, demonstrating minimal contamination of the RNA by tuber protein. While A260/A280 ratios of extracts from the other plant species were somewhat lower than those for potato (average = 1.56 and 1.80 for fresh and lyophilized samples, respectively), A260/A230 ratios averaged more than 2.0, and the RNA extracted from fresh and lyophilized samples of all species was intact, as demonstrated by denaturing agarose-formaldehyde gel electrophoresis. The protocol yielded RNA suitable for downstream molecular applications involving reverse transcription-polymerase chain reaction from all five species. Transcriptionally competent RNA was also recovered from lyophilized potato tuber tissue stored for 6 years (ambient temperature) by a simple modification to the protocol involving extraction in cold acetone. Lyophilization can thus be used to preserve RNA in high starch- and phenolic-containing plant tissues for studies on gene expression.

  1. Optimal Techniques for mRNA Extraction from Neonatal Salivary Supernatant

    PubMed Central

    Dietz, Jessica A.; Johnson, Kirby L.; Wick, Heather C.; Bianchi, Diana W.; Maron, Jill L.

    2011-01-01

    Background Gene expression profiling of the salivary supernatant is emerging as a new and important source of real-time, systemic, biological information. However, existing technologies prevent RNA extraction of small quantities found in neonatal salivary supernatant. Objective The aim of this study was to develop techniques to enhance extraction of cell-free RNA from neonatal salivary supernatant. Methods Two saliva samples (10–100 μl) were serially collected from newborns (36–41 weeks’ gestation) (n = 13) and stabilized. Total RNA was extracted from salivary supernatant with the use of two modified extraction techniques: Qiagen RNAprotect® Saliva Mini Kit (method 1) and the QIAamp Viral RNA Mini Kit (method 2). Quantitative RT-PCR amplification for GAPDH was performed on extracted salivary samples. Statistical analyses were performed on mean threshold cycle (Ct) levels to compare RNA yield from each protocol. Paired microarray analyses were made between neonatal whole saliva and supernatant (n = 3) to discern gene expression differences between these biolayers. Results mRNA was successfully extracted and amplified from all salivary supernatant samples. Extraction with method 2 yielded more RNA than with method 1 (p = 0.008). There was a 7.5% discordance between paired gene expression analyses for whole saliva and supernatant. Genes that were statistically significantly upregulated in supernatant highlighted 16 distinct biological functions not seen in whole saliva. Conversely, only two biological functions were unique to whole saliva. Conclusion Neonatal cell-free salivary supernatant mRNA may be readily extracted and utilized on downstream applications. These technical enhancements allow for further exploration of the diagnostic potential of the neonatal salivary transcriptome. PMID:21791940

  2. Automated endmember extraction for subpixel classification of multispectral and hyperspectral data

    NASA Astrophysics Data System (ADS)

    Shrivastava, Deepali; Kumar, Vinay; Sharma, Richa U.

    2016-04-01

    Most of the multispectral sensors acquire data in several broad wavelength bands and are capable of extracting different Land Cover features while hyperspectral sensors contain ample spectral data in narrow bandwidth (10- 20nm). The spectrally rich data enable the extraction of useful quantitative information from earth surface features. Endmembers are the pure spectral components extracted from the remote sensing datasets. Most approaches for Endmember extraction (EME) are manual and have been designed from a spectroscopic viewpoint, thus neglecting the spatial arrangement of the pixels. Therefore, EME techniques which can consider both spectral and spatial aspects are required to find more accurate Endmembers for Subpixel classification. Multispectral (EO-1 ALI and Landsat 8 OLI) and Hyperspectral (EO-1 Hyperion) datasets of Udaipur region, Rajasthan is used in this study. All the above mentioned datasets are preprocessed and converted to surface reflectance using Fast Line-of-sight Atmospheric Analysis of Spectral Hypercube (FLAASH). Further Automated Endmember extraction and Subpixel classification is carried out using Multiple Endmember Spectral Mixture Analysis (MESMA). Endmembers are selected from spectral libraries to be given as input to MESMA. To optimize these spectral libraries three techniques are deployed i.e. Count based Endmember selection (CoB), Endmember Average RMSE (EAR) and Minimum Average Spectral Angle (MASA) for endmember selection. Further identified endmembers are used for classifying multispectral and hyperspectral data using MESMA and SAM. It was observed from the obtained classified results that diverse features, spread over a pixel, which are spectrally same are well classified by MESMA whereas SAM was unable to do so.

  3. Automated multisyringe stir bar sorptive extraction using robust montmorillonite/epoxy-coated stir bars.

    PubMed

    Ghani, Milad; Saraji, Mohammad; Maya, Fernando; Cerdà, Víctor

    2016-05-06

    Herein we present a simple, rapid and low cost strategy for the preparation of robust stir bar coatings based on the combination of montmorillonite with epoxy resin. The composite stir bar was implemented in a novel automated multisyringe stir bar sorptive extraction system (MS-SBSE), and applied to the extraction of four chlorophenols (4-chlorophenol, 2,4-dichlorophenol, 2,4,6-trichlorophenol and pentachlorophenol) as model compounds, followed by high performance liquid chromatography-diode array detection. The different experimental parameters of the MS-SBSE, such as sample volume, selection of the desorption solvent, desorption volume, desorption time, sample solution pH, salt effect and extraction time were studied. Under the optimum conditions, the detection limits were between 0.02 and 0.34μgL(-1). Relative standard deviations (RSD) of the method for the analytes at 10μgL(-1) concentration level ranged from 3.5% to 4.1% (as intra-day RSD) and from 3.9% to 4.3% (as inter-day RSD at 50μgL(-1) concentration level). Batch-to-batch reproducibility for three different stir bars was 4.6-5.1%. The enrichment factors were between 30 and 49. In order to investigate the capability of the developed technique for real sample analysis, well water, wastewater and leachates from a solid waste treatment plant were satisfactorily analyzed.

  4. Knowledge-based automated feature extraction to categorize secondary digitized radiographs

    NASA Astrophysics Data System (ADS)

    Kohnen, Michael; Vogelsang, Frank; Wein, Berthold B.; Kilbinger, Markus W.; Guenther, Rolf W.; Weiler, Frank; Bredno, Joerg; Dahmen, Joerg

    2000-06-01

    An essential part of the IRMA-project (Image Retrieval in Medical Applications) is the categorization of digitized images into predefined classes using a combination of different independent features. To obtain an automated and content-based categorization, the following features are extracted from the image data: Fourier coefficients of normalized projections are computed to supply a scale- and translation-invariant description. Furthermore, histogram information and Co-occurrence matrices are calculated to supply information about the gray value distribution and textural information. But the key part of the feature extraction is the shape information of the objects represented by an Active Shape Model. The Active Shape Model supports various form variations given by a representative training set; we use one particular Active Shape Model for each image class. These different Active Shape Models are matched on preprocessed image data with a simulated annealing optimization. The different extracted features were chosen with regard to the different characteristics of the image content. They give a comprehensive description of image content using only few different features. Using this combination of different features for categorization results in a robust classification of image data, which is a basic step towards medical archives that allow retrieval results for queries of diagnostic relevance.

  5. Evaluation of automated urban surface water extraction from Sentinel-2A imagery using different water indices

    NASA Astrophysics Data System (ADS)

    Yang, Xiucheng; Chen, Li

    2017-04-01

    Urban surface water is characterized by complex surface continents and small size of water bodies, and the mapping of urban surface water is currently a challenging task. The moderate-resolution remote sensing satellites provide effective ways of monitoring surface water. This study conducts an exploratory evaluation on the performance of the newly available Sentinel-2A multispectral instrument (MSI) imagery for detecting urban surface water. An automatic framework that integrates pixel-level threshold adjustment and object-oriented segmentation is proposed. Based on the automated workflow, different combinations of visible, near infrared, and short-wave infrared bands in Sentinel-2 image via different water indices are first compared. Results show that object-level modified normalized difference water index (MNDWI with band 11) and automated water extraction index are feasible in urban surface water mapping for Sentinel-2 MSI imagery. Moreover, comparative results are obtained utilizing optimal MNDWI from Sentinel-2 and Landsat 8 images, respectively. Consequently, Sentinel-2 MSI achieves the kappa coefficient of 0.92, compared with that of 0.83 from Landsat 8 operational land imager.

  6. Investigation of automated feature extraction techniques for applications in cancer detection from multispectral histopathology images

    NASA Astrophysics Data System (ADS)

    Harvey, Neal R.; Levenson, Richard M.; Rimm, David L.

    2003-05-01

    Recent developments in imaging technology mean that it is now possible to obtain high-resolution histological image data at multiple wavelengths. This allows pathologists to image specimens over a full spectrum, thereby revealing (often subtle) distinctions between different types of tissue. With this type of data, the spectral content of the specimens, combined with quantitative spatial feature characterization may make it possible not only to identify the presence of an abnormality, but also to classify it accurately. However, such are the quantities and complexities of these data, that without new automated techniques to assist in the data analysis, the information contained in the data will remain inaccessible to those who need it. We investigate the application of a recently developed system for the automated analysis of multi-/hyper-spectral satellite image data to the problem of cancer detection from multispectral histopathology image data. The system provides a means for a human expert to provide training data simply by highlighting regions in an image using a computer mouse. Application of these feature extraction techniques to examples of both training and out-of-training-sample data demonstrate that these, as yet unoptimized, techniques already show promise in the discrimination between benign and malignant cells from a variety of samples.

  7. Online coupling of pressurized liquid extraction, solid-phase extraction and high-performance liquid chromatography for automated analysis of proanthocyanidins in malt.

    PubMed

    Papagiannopoulos, Menelaos; Zimmermann, Benno; Mellenthin, Annett; Krappe, Martin; Maio, Giovanni; Galensa, Rudolf

    2002-06-07

    A new instrumental setup for automated extraction of solid samples by online coupling of pressurized liquid extraction, automated SPE (solid-phase extraction) and HPLC is presented. From the extraction to the chromatogram no manual sample handling is required. The application to the determination of proanthocyanidins in malt reduces time and manual work to a minimum compared to former manual methods. Twenty samples can be processed within 24 h in respect to eight samples with the manual method. Using the features of the instrumental coupling, an optimized strategy for SPE of proanthocyanidins from natural samples was developed, requiring no evaporation step, using commercial cartridges and delivering concentrated eluates. The recovery of five main malt proanthocyanidins was 97%, with a reproducibility of 5%. This new instrumental coupling is thought to reduce time and costs along with improved results for a broad range of solid sample materials.

  8. RNA Extraction from Animal and Human's Cancerous Tissues: Does Tissue Matter?

    PubMed

    Samadani, Ali Akbar; Nikbakhsh, Novin; Fattahi, Sadegh; Pourbagher, Roghayeh; Aghajanpour Mir, Seyyed Mohsen; Mousavi Kani, Narges; Abedian, Zeinab; Akhavan-Niaki, Haleh

    2015-01-01

    The reliability of gene expression profiling, based technologies and methods to find transcriptional differences representative of the original samples is influenced by the quality of the extracted RNA. Hence, RNA extraction is the first step to investigate the gene expression and its function. Consequently, the quality of extracted RNA is really significant. Correspondingly, this research was accomplished to optimize the RNA extraction methods and compare the amounts of tissue or quality of tissue. Relatively, the cancerous tissue of human stomach in fresh and frozen conditions and also the mouse fresh tissue were studied. Some factors like the amount of samples, efficacy differences of diverse extraction buffers (TriPure, Trizol) and also the efficacy of b-mercaptoethanol were compared and investigated. The results indicated that the less amount (1-2 mg) compared to other amounts (2-5 mg, 5-15 mg) yielded the best quality and the RNA bands (5S, 18S, 28S) were observed perfectly. Relatively, comparing and measuring some kinds of buffers (Trizol, TriPure) indicated no difference in RNA extraction quality. The last investigated factor was the effect of b- mercaptoethanol which was used along with TriPure to remove the RNAse. Conclusively, no effective impression was observed.

  9. An effective method for extracting total RNA from Dioscorea opposita Thunb.

    PubMed

    Liao, C M; Li, J; Liu, X H; Zhang, Y S

    2014-01-21

    Dioscorea opposita Thunb., included in the genus Dioscorea of the family Dioscoreaceae, is an important herb with great edible and medicinal value. In this study, the total RNA from leaves of Lichuan Dioscorea opposita Thunb. was isolated by an improved Trizol method. The results showed that the RNA extracted by the improved Trizol method had good integrity, and the RNA could be used for down-stream molecular biology operations including reverse transcription-polymerase chain reaction.

  10. Prospective evaluation of a new automated nucleic acid extraction system using routine clinical respiratory specimens.

    PubMed

    Mengelle, C; Mansuy, J-M; Sandres-Sauné, K; Barthe, C; Boineau, J; Izopet, J

    2012-06-01

    The aim of the study was to evaluate the MagNA Pure 96™ nucleic acid extraction system using clinical respiratory specimens for identifying viruses by qualitative real-time PCR assays. Three extraction methods were tested, that is, the MagNA Pure LC™, the COBAS Ampliprep™, and the MagNA Pure 96™ with 10-fold dilutions of an influenza A(H1N1)pdm09 sample. Two hundred thirty-nine respiratory specimens, 35 throat swabs, 164 nasopharyngeal specimens, and 40 broncho-alveolar fluids, were extracted with the MagNA Pure 96™ and the COBAS Ampliprep™ instruments. Forty COBAS Ampliprep™ positive samples were also tested. Real-time PCRs were used to identify influenza A and influenza A(H1N1)pdm09, rhinovirus, enterovirus, adenovirus, varicella zoster virus, cytomegalovirus, and herpes simplex virus. Similar results were obtained on RNA extracted from dilutions of influenza A(H1N1)pdm09 with the three systems: the MagNA Pure LC™, the COBAS Ampliprep™, and the MagNA Pure 96™. Data from clinical respiratory specimens extracted with the MagNA Pure 96™ and COBAS Ampliprep™ instruments were in 98.5% in agreement (P < 0.0001) for influenza A and influenza A(H1N1)pdm09. Data for rhinovirus were in 97.3% agreement (P < 0.0001) and in 96.8% agreement for enterovirus. They were in 100% agreement for adenovirus. Data for cytomegalovirus and HSV1-2 were in 95.2% agreement (P < 0.0001). The MagNA Pure 96™ instrument is easy-to-use, reliable, and has a high throughput for extracting total nucleic acid from respiratory specimens. These extracts are suitable for molecular diagnosis with any type of real-time PCR assay.

  11. Plasma free metanephrine measurement using automated online solid-phase extraction HPLC tandem mass spectrometry.

    PubMed

    de Jong, Wilhelmina H A; Graham, Kendon S; van der Molen, Jan C; Links, Thera P; Morris, Michael R; Ross, H Alec; de Vries, Elisabeth G E; Kema, Ido P

    2007-09-01

    Quantification of plasma free metanephrine (MN) and normetanephrine (NMN) is considered to be the most accurate test for the clinical chemical diagnosis of pheochromocytoma and follow-up of pheochromocytoma patients. Current methods involve laborious, time-consuming, offline sample preparation, coupled with relatively nonspecific detection. Our aim was to develop a rapid, sensitive, and highly selective automated method for plasma free MNs in the nanomole per liter range. We used online solid-phase extraction coupled with HPLC-tandem mass spectrometric detection (XLC-MS/MS). Fifty microliters plasma equivalent was prepurified by automated online solid-phase extraction, using weak cation exchange cartridges. Chromatographic separation of the analytes and deuterated analogs was achieved by hydrophilic interaction chromatography. Mass spectrometric detection was performed in the multiple reaction monitoring mode using a quadrupole tandem mass spectrometer in positive electrospray ionization mode. Total run-time including sample cleanup was 8 min. Intra- and interassay analytical variation (CV) varied from 2.0% to 4.7% and 1.6% to 13.5%, respectively, whereas biological intra- and interday variation ranged from 9.4% to 45.0% and 8.4% to 23.2%. Linearity in the 0 to 20 nmol/L calibration range was excellent (R(2) > 0.99). For all compounds, recoveries ranged from 74.5% to 99.6%, and detection limits were <0.10 nmol/L. Reference intervals for 120 healthy adults were 0.07 to 0.33 nmol/L (MN), 0.23 to 1.07 nmol/L (NMN), and <0.17 nmol/L (3-methoxytyramine). This automated high-throughput XLC-MS/MS method for the measurement of plasma free MNs is precise and linear, with short analysis time and low variable costs. The method is attractive for routine diagnosis of pheochromocytoma because of its high analytical sensitivity, the analytical power of MS/MS, and the high diagnostic accuracy of free MNs.

  12. Automated extraction of the cortical sulci based on a supervised learning approach.

    PubMed

    Tu, Zhuowen; Zheng, Songfeng; Yuille, Alan L; Reiss, Allan L; Dutton, Rebecca A; Lee, Agatha D; Galaburda, Albert M; Dinov, Ivo; Thompson, Paul M; Toga, Arthur W

    2007-04-01

    It is important to detect and extract the major cortical sulci from brain images, but manually annotating these sulci is a time-consuming task and requires the labeler to follow complex protocols. This paper proposes a learning-based algorithm for automated extraction of the major cortical sulci from magnetic resonance imaging (MRI) volumes and cortical surfaces. Unlike alternative methods for detecting the major cortical sulci, which use a small number of predefined rules based on properties of the cortical surface such as the mean curvature, our approach learns a discriminative model using the probabilistic boosting tree algorithm (PBT). PBT is a supervised learning approach which selects and combines hundreds of features at different scales, such as curvatures, gradients and shape index. Our method can be applied to either MRI volumes or cortical surfaces. It first outputs a probability map which indicates how likely each voxel lies on a major sulcal curve. Next, it applies dynamic programming to extract the best curve based on the probability map and a shape prior. The algorithm has almost no parameters to tune for extracting different major sulci. It is very fast (it runs in under 1 min per sulcus including the time to compute the discriminative models) due to efficient implementation of the features (e.g., using the integral volume to rapidly compute the responses of 3-D Haar filters). Because the algorithm can be applied to MRI volumes directly, there is no need to perform preprocessing such as tissue segmentation or mapping to a canonical space. The learning aspect of our approach makes the system very flexible and general. For illustration, we use volumes of the right hemisphere with several major cortical sulci manually labeled. The algorithm is tested on two groups of data, including some brains from patients with Williams Syndrome, and the results are very encouraging.

  13. RNA extraction method is crucial for human papillomavirus E6/E7 oncogenes detection.

    PubMed

    Fontecha, Nerea; Nieto, Maria Carmen; Andía, Daniel; Cisterna, Ramón; Basaras, Miren

    2017-03-09

    Human papillomavirus (HPV) DNA testing plays a main role in the management of cervical cancer, however to improve the specificity in cervical screening, there is a need to develop and validate different approaches that can identify women at risk for progressive disease. Nowadays, mRNA expression of viral E6 and E7 HPV oncogenes stands up as a potential biomarker to improve cervical screening. We aimed to validate a method for RNA extraction, detect HPV mRNA expression and, assess the relationship between E6/E7 mRNA expression and pathology of patients' lesions and progression. This study included 50 specimens that had been previously genotyped as HPV16, 18, 31, 33 and/or 45. Cervical swabs were extracted with three different RNA extraction methods -Nuclisens manual extraction kit (bioMérieux), High Pure Viral RNA Kit (Roche) and RNeasy Plus Mini kit (Qiagen)-, and mRNA was detected with NucliSens EasyQ HPV version 1 test (bioMérieux) afterwards. Association of oncogene expression with pathology and lesion progression was analyzed for each extraction method. E6/E7 mRNA positivity rate was higher in samples analyzed with bioMérieux (62%), followed by Roche (24%) and Qiagen (6%). Women with lesions and lesion progression showed a higher prevalence of viral RNA expression than women that had not lesions or with lesion persistence. While bioMérieux revealed a higher sensitivity (77.27%), Roche presented a higher PPV (75%) and an increased specificity (89.28%). Extraction methods based on magnetic beads provided better RNA yield than those based in columns. Both Nuclisens manual extraction kit (bioMérieux) and High Pure Viral RNA Kit (Roche) seemed to be adequate for E6/E7 mRNA detection. However, none of them revealed both high sensitivity and specificity values. Further studies are needed to obtain and validate a standard gold method for RNA expression detection, to be included as part of the routine cervical screening program.

  14. Urinary extracellular vesicles for RNA extraction: optimization of a protocol devoid of prokaryote contamination

    PubMed Central

    Tataruch-Weinert, Dorota; Musante, Luca; Kretz, Oliver; Holthofer, Harry

    2016-01-01

    Background Urinary extracellular vesicles (UEVs) represent an ideal platform for biomarker discovery. They carry different types of RNA species, and reported profile discrepancies related to the presence/absence of 18s and 28s rRNA remain controversial. Moreover, sufficient urinary RNA yields and respective quality RNA profiles are still to be fully established. Methods UEVs were enriched by hydrostatic filtration dialysis, and RNA content was extracted using 7 different commercially available techniques. RNA quantity was assessed using spectrophotometry and fluorometry, whilst RNA quality was determined by capillary electrophoresis. Results The presence of prokaryotic transcriptome was stressed when cellular RNA, as a control, was spiked into the UEVs samples before RNA extraction. The presence of bacteria in hydrostatic filtration dialysis above 1,000 kDa molecular weight cut-off and in crude urine was confirmed with growth media plates. The efficiency in removing urinary bacteria was evaluated by differential centrifugation, filtration (0.22 µm filters) and chemical pretreatment (water purification tablet). For volumes of urine >200 ml, the chemical treatment provides ease of handling without affecting vesicle integrity, protein and RNA profiles. This protocol was selected to enrich RNA with 7 methods, and its respective quality and quantity were assessed. The results were given as follows: (a) Fluorometry gave more repeatability and reproducibility than spectrophotometry to assess the RNA yields, (b) UEVs were enriched with small RNA, (c) Ribosomal RNA peaks were not observed for any RNA extraction method used and (d) RNA yield was higher for column-based method designed for urinary exosome, whilst the highest relative microRNA presence was obtained using TRIzol method. Conclusion Our results show that the presence of bacteria can lead to misidentification in the electrophoresis peaks. Fluorometry is more reliable than spectrophotometry. RNA isolation method

  15. High-quality RNA extraction from small cardamom tissues rich in polysaccharides and polyphenols.

    PubMed

    Nadiya, Fasiludeen; Anjali, Narayanannair; Gangaprasad, Appukuttannair; Sabu, Kalluvettankuzhy Krishnannair

    2015-09-15

    Due to the presence of a diverse array of metabolites, no standard method of RNA isolation is available for plants. We noted that polysaccharide and polyphenol contents of cardamom tissues critically hinder the RNA extraction procedure. Hence, we attempted several methods for obtaining intact mRNA and small RNA from various cardamom tissues. It was found that protocols involving a combination of commercial kits and conventional CTAB (cetyl trimethylammonium bromide) methods yielded RNA with good purity, higher yield, and good integrity. The total RNA isolated through this approach was found to be amenable for transcriptome and small RNA analysis through next-generation sequencing platforms. Copyright © 2015 Elsevier Inc. All rights reserved.

  16. [The development of kit for hybridization extraction of DNA and RNA of agents of hemotransmissive infections from serum and blood plasma].

    PubMed

    Netesova, E S; Bragin, A G; Glushkov, S A; Prasolova, M A; Dymshits, G M; Kondrushin, E V; Podymova, A S; Sandyreva, G M

    2014-10-01

    To decrease dependence of effectiveness of isolation of nucleic acids of composition and amount of applied sample a kit was developed for hybridization extraction of DNA HBV RNA HCV and RNA HIV from blood serum in two formats--using up to 250 mkl and up to 1 ml of sample. This kit, in complex with kits for detection using polymerase chain reaction technique in real-time, forms a test characterized by high analytical sensitivity i.e. HBV50 copies per ml, HCV37.5 copies per ml, HIV 13 copies per ml. The developed kit for extraction of target nucleic acids permits to get rid of total DNA and inhibited effect of heparin. It can be adapted for application wit factors B and automated stations of sample preparation.

  17. Validation of the Total Visual Acuity Extraction Algorithm (TOVA) for Automated Extraction of Visual Acuity Data From Free Text, Unstructured Clinical Records

    PubMed Central

    Baughman, Douglas M.; Su, Grace L.; Tsui, Irena; Lee, Cecilia S.; Lee, Aaron Y.

    2017-01-01

    Purpose With increasing volumes of electronic health record data, algorithm-driven extraction may aid manual extraction. Visual acuity often is extracted manually in vision research. The total visual acuity extraction algorithm (TOVA) is presented and validated for automated extraction of visual acuity from free text, unstructured clinical notes. Methods Consecutive inpatient ophthalmology notes over an 8-year period from the University of Washington healthcare system in Seattle, WA were used for validation of TOVA. The total visual acuity extraction algorithm applied natural language processing to recognize Snellen visual acuity in free text notes and assign laterality. The best corrected measurement was determined for each eye and converted to logMAR. The algorithm was validated against manual extraction of a subset of notes. Results A total of 6266 clinical records were obtained giving 12,452 data points. In a subset of 644 validated notes, comparison of manually extracted data versus TOVA output showed 95% concordance. Interrater reliability testing gave κ statistics of 0.94 (95% confidence interval [CI], 0.89–0.99), 0.96 (95% CI, 0.94–0.98), 0.95 (95% CI, 0.92–0.98), and 0.94 (95% CI, 0.90–0.98) for acuity numerators, denominators, adjustments, and signs, respectively. Pearson correlation coefficient was 0.983. Linear regression showed an R2 of 0.966 (P < 0.0001). Conclusions The total visual acuity extraction algorithm is a novel tool for extraction of visual acuity from free text, unstructured clinical notes and provides an open source method of data extraction. Translational Relevance Automated visual acuity extraction through natural language processing can be a valuable tool for data extraction from free text ophthalmology notes. PMID:28299240

  18. BLINKER: Automated Extraction of Ocular Indices from EEG Enabling Large-Scale Analysis.

    PubMed

    Kleifges, Kelly; Bigdely-Shamlo, Nima; Kerick, Scott E; Robbins, Kay A

    2017-01-01

    Electroencephalography (EEG) offers a platform for studying the relationships between behavioral measures, such as blink rate and duration, with neural correlates of fatigue and attention, such as theta and alpha band power. Further, the existence of EEG studies covering a variety of subjects and tasks provides opportunities for the community to better characterize variability of these measures across tasks and subjects. We have implemented an automated pipeline (BLINKER) for extracting ocular indices such as blink rate, blink duration, and blink velocity-amplitude ratios from EEG channels, EOG channels, and/or independent components (ICs). To illustrate the use of our approach, we have applied the pipeline to a large corpus of EEG data (comprising more than 2000 datasets acquired at eight different laboratories) in order to characterize variability of certain ocular indicators across subjects. We also investigate dependence of ocular indices on task in a shooter study. We have implemented our algorithms in a freely available MATLAB toolbox called BLINKER. The toolbox, which is easy to use and can be applied to collections of data without user intervention, can automatically discover which channels or ICs capture blinks. The tools extract blinks, calculate common ocular indices, generate a report for each dataset, dump labeled images of the individual blinks, and provide summary statistics across collections. Users can run BLINKER as a script or as a plugin for EEGLAB. The toolbox is available at https://github.com/VisLab/EEG-Blinks. User documentation and examples appear at http://vislab.github.io/EEG-Blinks/.

  19. Automated data extraction from in situ protein stable isotope probing studies

    SciTech Connect

    Slysz, Gordon W.; Steinke, Laurey A.; Ward, David M.; Klatt, Christian G.; Clauss, Therese RW; Purvine, Samuel O.; Payne, Samuel H.; Anderson, Gordon A.; Smith, Richard D.; Lipton, Mary S.

    2014-01-27

    Protein stable isotope probing (protein-SIP) has strong potential for revealing key metabolizing taxa in complex microbial communities. While most protein-SIP work to date has been performed under controlled laboratory conditions to allow extensive isotope labeling of the target organism, a key application will be in situ studies of microbial communities under conditions that result in small degrees of partial labeling. One hurdle restricting large scale in situ protein-SIP studies is the lack of algorithms and software for automated data processing of the massive data sets resulting from such studies. In response, we developed Stable Isotope Probing Protein Extraction Resources software (SIPPER) and applied it for large scale extraction and visualization of data from short term (3 h) protein-SIP experiments performed in situ on Yellowstone phototrophic bacterial mats. Several metrics incorporated into the software allow it to support exhaustive analysis of the complex composite isotopic envelope observed as a result of low amounts of partial label incorporation. SIPPER also enables the detection of labeled molecular species without the need for any prior identification.

  20. BLINKER: Automated Extraction of Ocular Indices from EEG Enabling Large-Scale Analysis

    PubMed Central

    Kleifges, Kelly; Bigdely-Shamlo, Nima; Kerick, Scott E.; Robbins, Kay A.

    2017-01-01

    Electroencephalography (EEG) offers a platform for studying the relationships between behavioral measures, such as blink rate and duration, with neural correlates of fatigue and attention, such as theta and alpha band power. Further, the existence of EEG studies covering a variety of subjects and tasks provides opportunities for the community to better characterize variability of these measures across tasks and subjects. We have implemented an automated pipeline (BLINKER) for extracting ocular indices such as blink rate, blink duration, and blink velocity-amplitude ratios from EEG channels, EOG channels, and/or independent components (ICs). To illustrate the use of our approach, we have applied the pipeline to a large corpus of EEG data (comprising more than 2000 datasets acquired at eight different laboratories) in order to characterize variability of certain ocular indicators across subjects. We also investigate dependence of ocular indices on task in a shooter study. We have implemented our algorithms in a freely available MATLAB toolbox called BLINKER. The toolbox, which is easy to use and can be applied to collections of data without user intervention, can automatically discover which channels or ICs capture blinks. The tools extract blinks, calculate common ocular indices, generate a report for each dataset, dump labeled images of the individual blinks, and provide summary statistics across collections. Users can run BLINKER as a script or as a plugin for EEGLAB. The toolbox is available at https://github.com/VisLab/EEG-Blinks. User documentation and examples appear at http://vislab.github.io/EEG-Blinks/. PMID:28217081

  1. Automated data extraction from in situ protein-stable isotope probing studies.

    PubMed

    Slysz, Gordon W; Steinke, Laurey; Ward, David M; Klatt, Christian G; Clauss, Therese R W; Purvine, Samuel O; Payne, Samuel H; Anderson, Gordon A; Smith, Richard D; Lipton, Mary S

    2014-03-07

    Protein-stable isotope probing (protein-SIP) has strong potential for revealing key metabolizing taxa in complex microbial communities. While most protein-SIP work to date has been performed under controlled laboratory conditions to allow extensive isotope labeling of the target organism(s), a key application will be in situ studies of microbial communities for short periods of time under natural conditions that result in small degrees of partial labeling. One hurdle restricting large-scale in situ protein-SIP studies is the lack of algorithms and software for automated data processing of the massive data sets resulting from such studies. In response, we developed Stable Isotope Probing Protein Extraction Resources software (SIPPER) and applied it for large-scale extraction and visualization of data from short-term (3 h) protein-SIP experiments performed in situ on phototrophic bacterial mats isolated from Yellowstone National Park. Several metrics incorporated into the software allow it to support exhaustive analysis of the complex composite isotopic envelope observed as a result of low amounts of partial label incorporation. SIPPER also enables the detection of labeled molecular species without the need for any prior identification.

  2. Deep Learning for Automated Extraction of Primary Sites from Cancer Pathology Reports.

    PubMed

    Qiu, John; Yoon, Hong-Jun; Fearn, Paul A; Tourassi, Georgia D

    2017-05-03

    for cancer registries which process high volumes of free-text reports annually. Information extraction and coding is a manual, labor-intensive process. In this study we investigated deep learning and a convolutional neural network (CNN), for extracting ICDO- 3 topographic codes from a corpus of breast and lung cancer pathology reports. We performed two experiments, using a CNN and a more conventional term frequency vector approach, to assess the effects of class prevalence and inter-class transfer learning. The experiments were based on a set of 942 pathology reports with human expert annotations as the gold standard. CNN performance was compared against a more conventional term frequency vector space approach. We observed that the deep learning models consistently outperformed the conventional approaches in the class prevalence experiment, resulting in micro and macro-F score increases of up to 0.132 and 0.226 respectively when class labels were well populated. Specifically, the best performing CNN achieved a micro-F score of 0.722 over 12 ICD-O-3 topography codes. Transfer learning provided a consistent but modest performance boost for the deep learning methods but trends were contingent on CNN method and cancer site. These encouraging results demonstrate the potential of deep learning for automated abstraction of pathology reports.

  3. Rapid and automated sample preparation for nucleic acid extraction on a microfluidic CD (compact disk)

    NASA Astrophysics Data System (ADS)

    Kim, Jitae; Kido, Horacio; Zoval, Jim V.; Gagné, Dominic; Peytavi, Régis; Picard, François J.; Bastien, Martine; Boissinot, Maurice; Bergeron, Michel G.; Madou, Marc J.

    2006-01-01

    Rapid and automated preparation of PCR (polymerase chain reaction)-ready genomic DNA was demonstrated on a multiplexed CD (compact disk) platform by using hard-to-lyse bacterial spores. Cell disruption is carried out while beadcell suspensions are pushed back and forth in center-tapered lysing chambers by angular oscillation of the disk - keystone effect. During this lysis period, the cell suspensions are securely held within the lysing chambers by heatactivated wax valves. Upon application of a remote heat to the disk in motion, the wax valves release lysate solutions into centrifuge chambers where cell debris are separated by an elevated rotation of the disk. Only debris-free DNA extract is then transferred to collection chambers by capillary-assisted siphon and collected for heating that inactivates PCR inhibitors. Lysing capacity was evaluated using a real-time PCR assay to monitor the efficiency of Bacillus globigii spore lysis. PCR analysis showed that 5 minutes' CD lysis run gave spore lysis efficiency similar to that obtained with a popular commercial DNA extraction kit (i.e., IDI-lysis kit from GeneOhm Sciences Inc.) which is highly efficient for microbial cell and spore lysis. This work will contribute to the development of an integrated CD-based assay for rapid diagnosis of infectious diseases.

  4. Simultaneous extraction of high-quality RNA and DNA from small tissue samples.

    PubMed

    Triant, Deborah A; Whitehead, Andrew

    2009-01-01

    Purification of high-quality DNA and RNA from a single sample is becoming increasingly important for studies seeking both genomic and transcriptomic data. We compare different methods for isolating DNA and RNA from fish embryos (Gulf killifish; Fundulus grandis) and describe an optimal technique to extract high-quality DNA and RNA from a single embryo. The optimal method utilizes a chaotropic buffer and spin column technology. From embryos weighing approximately 4 mg, we were able to isolate an average of 6.1 microg of DNA and 1.1 microg of RNA per sample. Relative amounts of DNA and RNA can be adjusted as needed per study. Although these extraction trials were conducted on fish embryos, they can be potentially applied to small samples that typically do not yield high concentrations of nucleic acids.

  5. Managing expectations: assessment of chemistry databases generated by automated extraction of chemical structures from patents.

    PubMed

    Senger, Stefan; Bartek, Luca; Papadatos, George; Gaulton, Anna

    2015-12-01

    First public disclosure of new chemical entities often takes place in patents, which makes them an important source of information. However, with an ever increasing number of patent applications, manual processing and curation on such a large scale becomes even more challenging. An alternative approach better suited for this large corpus of documents is the automated extraction of chemical structures. A number of patent chemistry databases generated by using the latter approach are now available but little is known that can help to manage expectations when using them. This study aims to address this by comparing two such freely available sources, SureChEMBL and IBM SIIP (IBM Strategic Intellectual Property Insight Platform), with manually curated commercial databases. When looking at the percentage of chemical structures successfully extracted from a set of patents, using SciFinder as our reference, 59 and 51 % were also found in our comparison in SureChEMBL and IBM SIIP, respectively. When performing this comparison with compounds as starting point, i.e. establishing if for a list of compounds the databases provide the links between chemical structures and patents they appear in, we obtained similar results. SureChEMBL and IBM SIIP found 62 and 59 %, respectively, of the compound-patent pairs obtained from Reaxys. In our comparison of automatically generated vs. manually curated patent chemistry databases, the former successfully provided approximately 60 % of links between chemical structure and patents. It needs to be stressed that only a very limited number of patents and compound-patent pairs were used for our comparison. Nevertheless, our results will hopefully help to manage expectations of users of patent chemistry databases of this type and provide a useful framework for more studies like ours as well as guide future developments of the workflows used for the automated extraction of chemical structures from patents. The challenges we have encountered

  6. Streamlining DNA Barcoding Protocols: Automated DNA Extraction and a New cox1 Primer in Arachnid Systematics

    PubMed Central

    Vidergar, Nina; Toplak, Nataša; Kuntner, Matjaž

    2014-01-01

    Background DNA barcoding is a popular tool in taxonomic and phylogenetic studies, but for most animal lineages protocols for obtaining the barcoding sequences—mitochondrial cytochrome C oxidase subunit I (cox1 AKA CO1)—are not standardized. Our aim was to explore an optimal strategy for arachnids, focusing on the species-richest lineage, spiders by (1) improving an automated DNA extraction protocol, (2) testing the performance of commonly used primer combinations, and (3) developing a new cox1 primer suitable for more efficient alignment and phylogenetic analyses. Methodology We used exemplars of 15 species from all major spider clades, processed a range of spider tissues of varying size and quality, optimized genomic DNA extraction using the MagMAX Express magnetic particle processor—an automated high throughput DNA extraction system—and tested cox1 amplification protocols emphasizing the standard barcoding region using ten routinely employed primer pairs. Results The best results were obtained with the commonly used Folmer primers (LCO1490/HCO2198) that capture the standard barcode region, and with the C1-J-2183/C1-N-2776 primer pair that amplifies its extension. However, C1-J-2183 is designed too close to HCO2198 for well-interpreted, continuous sequence data, and in practice the resulting sequences from the two primer pairs rarely overlap. We therefore designed a new forward primer C1-J-2123 60 base pairs upstream of the C1-J-2183 binding site. The success rate of this new primer (93%) matched that of C1-J-2183. Conclusions The use of C1-J-2123 allows full, indel-free overlap of sequences obtained with the standard Folmer primers and with C1-J-2123 primer pair. Our preliminary tests suggest that in addition to spiders, C1-J-2123 will also perform in other arachnids and several other invertebrates. We provide optimal PCR protocols for these primer sets, and recommend using them for systematic efforts beyond DNA barcoding. PMID:25415202

  7. Design and self-assembly of siRNA-functionalized RNA nanoparticles for use in automated nanomedicine

    PubMed Central

    Afonin, Kirill A; Grabow, Wade W; Walker, Faye M; Bindewald, Eckart; Dobrovolskaia, Marina A; Shapiro, Bruce A; Jaeger, Luc

    2012-01-01

    Individual genes can be targeted with siRNAs. The use of nucleic acid nanoparticles (NPs) is a convenient method for delivering combinations of specific siRNAs in an organized and programmable manner. We present three assembly protocols to produce two different types of RNA self-assembling functional NPs using processes that are fully automatable. These NPs are engineered based on two complementary nanoscaffold designs (nanoring and nanocube), which serve as carriers of multiple siRNAs. The NPs are functionalized by the extension of up to six scaffold strands with siRNA duplexes. The assembly protocols yield functionalized RNA NPs, and we show that they interact in vitro with human recombinant Dicer to produce siRNAs. Our design strategies allow for fast, economical and easily controlled production of endotoxin-free therapeutic RNA NPs that are suitable for preclinical development. PMID:22134126

  8. Comparison and optimization of methods for the simultaneous extraction of DNA, RNA, proteins, and metabolites.

    PubMed

    Vorreiter, Fränze; Richter, Silke; Peter, Michel; Baumann, Sven; von Bergen, Martin; Tomm, Janina M

    2016-09-01

    The challenge of performing a time-resolved comprehensive analysis of molecular systems has led to the quest to optimize extraction methods. When the size of a biological sample is limited, there is demand for the simultaneous extraction of molecules representing the four areas of "omics": genomics, transcriptomics, proteomics, and metabolomics. Here we optimized a protocol for the simultaneous extraction of DNA, RNA, proteins, and metabolites and compared it with two existing protocols. Our optimization comprised the addition of a methanol/chloroform metabolite purification before the separation of DNA/RNA and proteins. Extracted DNA, RNA, proteins, and metabolites were quantitatively and/or qualitatively analyzed. Of the three methods, only the newly developed protocol yielded all biomolecule classes of adequate quantity and quality.

  9. [Modified TRIzol method for RNA and DNA co-extraction from blood].

    PubMed

    Qin, Juan-Juan; Lu, Zhi-Yong; Jiao, Zhang-Ping; Zhu, Xiao-Jun; Wang, Ying-Xi; Tang, Hui

    2013-06-01

    To establish a new method for RNA and DNA co-extraction from the same sample by TRIzol reagent. After the aqueous phase which contained total RNA was removed by traditional TRIzol method, the values of pH of the interphase phase and organic phase were adjusted. The DNA was precipitated with ethanol and purified with DNA IQ system. The purified DNA was measured in quality and quantity. As the template, it was amplified and typed by PCR-STR. The data was compared with that extracted by traditional TRIzol method. The DNA extracted by this modified method showed a better result of quality and quantity than that by traditional TRIzol method and a good STR typing. The modified TRIzol method is advisable and reliable to simultaneously extract both DNA and RNA from the same sample. It could be used for individual identification and paternity testing to satisfy the need of forensic science.

  10. Automated fast extraction of nitrated polycyclic aromatic hydrocarbons from soil by focused microwave-assisted Soxhlet extraction prior to gas chromatography--electron-capture detection.

    PubMed

    Priego-Capote, F; Luque-García, J L; Luque de Castro, M D

    2003-04-25

    An approach for the automated fast extraction of nitrated polycyclic aromatic hydrocarbons (nitroPAHs) from soil, using a focused microwave-assisted Soxhlet extractor, is proposed. The main factors affecting the extraction efficiency (namely: irradiation power, irradiation time, number of cycles and extractant volume) were optimised by using experimental design methodology. The reduction of the nitro-PAHs to amino-PAHs and the derivatisation of the reduced analytes with heptafluorobutyric anhydride was mandatory prior to the separation-determination step by gas chromatography--electron-capture detection. The proposed approach has allowed the extraction of these pollutants from spiked and "real" contaminated soils with extraction efficiencies similar to those provided by the US Environmental Protection Agency methods 3540-8091, but with a drastic reduction in both the extraction time and sample handling, and using less organic solvent, as 75-85% of it was recycled.

  11. Development of automated extraction method of biliary tract from abdominal CT volumes based on local intensity structure analysis

    NASA Astrophysics Data System (ADS)

    Koga, Kusuto; Hayashi, Yuichiro; Hirose, Tomoaki; Oda, Masahiro; Kitasaka, Takayuki; Igami, Tsuyoshi; Nagino, Masato; Mori, Kensaku

    2014-03-01

    In this paper, we propose an automated biliary tract extraction method from abdominal CT volumes. The biliary tract is the path by which bile is transported from liver to the duodenum. No extraction method have been reported for the automated extraction of the biliary tract from common contrast CT volumes. Our method consists of three steps including: (1) extraction of extrahepatic bile duct (EHBD) candidate regions, (2) extraction of intrahepatic bile duct (IHBD) candidate regions, and (3) combination of these candidate regions. The IHBD has linear structures and intensities of the IHBD are low in CT volumes. We use a dark linear structure enhancement (DLSE) filter based on a local intensity structure analysis method using the eigenvalues of the Hessian matrix for the IHBD candidate region extraction. The EHBD region is extracted using a thresholding process and a connected component analysis. In the combination process, we connect the IHBD candidate regions to each EHBD candidate region and select a bile duct region from the connected candidate regions. We applied the proposed method to 22 cases of CT volumes. An average Dice coefficient of extraction result was 66.7%.

  12. Extraction and fractionation of RNA and DNA from single cells using selective lysing and isotachophoresis

    NASA Astrophysics Data System (ADS)

    Shintaku, Hirofumi; Santiago, Juan G.

    2015-03-01

    Single cell analyses of RNA and DNA are crucial to understanding the heterogeneity of cell populations. The numbers of approaches to single cells analyses are expanding, but sequence specific measurements of nucleic acids have been mostly limited to studies of either DNA or RNA, and not both. This remains a challenge as RNA and DNA have very similar physical and biochemical properties, and cross-contamination with each other can introduce false positive results. We present an electrokinetic technique which creates the opportunity to fractionate and deliver cytoplasmic RNA and genomic DNA to independent downstream analyses. Our technique uses an on-chip system that enables selective lysing of cytoplasmic membrane, extraction of RNA (away from genomic DNA and nucleus), focusing, absolute quantification of cytoplasmic RNA mass. The absolute RNA mass quantification is performed using fluorescence observation without enzymatic amplification in < 5 min. The cell nucleus is left intact and the relative genomic DNA amount in the nucleus can be measured. We demonstrate the technique using single mouse B lymphocyte cells, for which we extracted an average of 14.1 pg total cytoplasmic RNA per cell. We also demonstrate correlation analysis between the absolute amount of cytoplasmic RNA and relative amount of genomic DNA, showing heterogeneity associated with cell cycle.

  13. Automated Detection and Extraction of Coronal Dimmings from SDO/AIA Data

    NASA Astrophysics Data System (ADS)

    Wills-Davey, Meredith; Attrill, G. D. R.

    2009-05-01

    The sheer volume of data anticipated from the Solar Dynamics Observatory/Atmospheric Imaging Assembly (SDO/AIA) highlights the necessity for the development of automatic detection methods for various types of solar activity. Initially recognized in the 1970s, it is now well established that coronal dimmings are closely associated with coronal mass ejections (CMEs), and are particularly recognized as a reliable indicator of front-side (halo) CMEs, which can be difficult to detect in white-light coronagraph data. Existing work demonstrates that (i) estimates of the dimming volume can be related to the CME mass, (ii) the spatial extent of coronal dimmings gives information regarding the angular extent of the associated CME, (iii) measurement of the magnetic flux in dimming regions can be compared to that contained in modeled magnetic clouds, (iv) the evolution of coronal dimmings gives information about the development of the CME post-eruption, and (v) the distribution of the dimmings and their order of formation can be used to derive an understanding of the CME's early evolution. An automated coronal dimming region detection and extraction algorithm removes visual observer bias from determination of physical quantities described above. This allows reproducible, quantifiable results to be mined from very large datasets. The information derived may facilitate more reliable early space weather detection, as well as offering the potential for conducting large-sample studies focused on determining the geoeffectiveness of CMEs, coupled with analysis of their associated coronal dimmings. We present examples of dimming events extracted using our algorithm from existing EUV data, demonstrating the potential for the anticipated application to SDO/AIA data. Metadata returned by our algorithm include: location, area, volume, mass and dynamics of coronal dimmings. As well as running on historic datasets, this algorithm is capable of detecting and extracting coronal dimmings in

  14. Automated feature extraction for retinal vascular biometry in zebrafish using OCT angiography

    NASA Astrophysics Data System (ADS)

    Bozic, Ivan; Rao, Gopikrishna M.; Desai, Vineet; Tao, Yuankai K.

    2017-02-01

    Zebrafish have been identified as an ideal model for angiogenesis because of anatomical and functional similarities with other vertebrates. The scale and complexity of zebrafish assays are limited by the need to manually treat and serially screen animals, and recent technological advances have focused on automation and improving throughput. Here, we use optical coherence tomography (OCT) and OCT angiography (OCT-A) to perform noninvasive, in vivo imaging of retinal vasculature in zebrafish. OCT-A summed voxel projections were low pass filtered and skeletonized to create an en face vascular map prior to connectivity analysis. Vascular segmentation was referenced to the optic nerve head (ONH), which was identified by automatically segmenting the retinal pigment epithelium boundary on the OCT structural volume. The first vessel branch generation was identified as skeleton segments with branch points closest to the ONH, and subsequent generations were found iteratively by expanding the search space outwards from the ONH. Biometric parameters, including length, curvature, and branch angle of each vessel segment were calculated and grouped by branch generation. Despite manual handling and alignment of each animal over multiple time points, we observe distinct qualitative patterns that enable unique identification of each eye from individual animals. We believe this OCT-based retinal biometry method can be applied for automated animal identification and handling in high-throughput organism-level pharmacological assays and genetic screens. In addition, these extracted features may enable high-resolution quantification of longitudinal vascular changes as a method for studying zebrafish models of retinal neovascularization and vascular remodeling.

  15. Automated extraction and validation of children's gait parameters with the Kinect.

    PubMed

    Motiian, Saeid; Pergami, Paola; Guffey, Keegan; Mancinelli, Corrie A; Doretto, Gianfranco

    2015-12-02

    Gait analysis for therapy regimen prescription and monitoring requires patients to physically access clinics with specialized equipment. The timely availability of such infrastructure at the right frequency is especially important for small children. Besides being very costly, this is a challenge for many children living in rural areas. This is why this work develops a low-cost, portable, and automated approach for in-home gait analysis, based on the Microsoft Kinect. A robust and efficient method for extracting gait parameters is introduced, which copes with the high variability of noisy Kinect skeleton tracking data experienced across the population of young children. This is achieved by temporally segmenting the data with an approach based on coupling a probabilistic matching of stride template models, learned offline, with the estimation of their global and local temporal scaling. A preliminary study conducted on healthy children between 2 and 4 years of age is performed to analyze the accuracy, precision, repeatability, and concurrent validity of the proposed method against the GAITRite when measuring several spatial and temporal children's gait parameters. The method has excellent accuracy and good precision, with segmenting temporal sequences of body joint locations into stride and step cycles. Also, the spatial and temporal gait parameters, estimated automatically, exhibit good concurrent validity with those provided by the GAITRite, as well as very good repeatability. In particular, on a range of nine gait parameters, the relative and absolute agreements were found to be good and excellent, and the overall agreements were found to be good and moderate. This work enables and validates the automated use of the Kinect for children's gait analysis in healthy subjects. In particular, the approach makes a step forward towards developing a low-cost, portable, parent-operated in-home tool for clinicians assisting young children.

  16. Californian demonstration and validation of automated agricultural field extraction from multi-temporal Landsat data

    NASA Astrophysics Data System (ADS)

    Yan, L.; Roy, D. P.

    2013-12-01

    The spatial distribution of agricultural fields is a fundamental description of rural landscapes and the location and extent of fields is important to establish the area of land utilized for agricultural yield prediction, resource allocation, and for economic planning. To date, field objects have not been extracted from satellite data over large areas because of computational constraints and because consistently processed appropriate resolution data have not been available or affordable. We present a fully automated computational methodology to extract agricultural fields from 30m Web Enabled Landsat data (WELD) time series and results for approximately 250,000 square kilometers (eleven 150 x 150 km WELD tiles) encompassing all the major agricultural areas of California. The extracted fields, including rectangular, circular, and irregularly shaped fields, are evaluated by comparison with manually interpreted Landsat field objects. Validation results are presented in terms of standard confusion matrix accuracy measures and also the degree of field object over-segmentation, under-segmentation, fragmentation and shape distortion. The apparent success of the presented field extraction methodology is due to several factors. First, the use of multi-temporal Landsat data, as opposed to single Landsat acquisitions, that enables crop rotations and inter-annual variability in the state of the vegetation to be accommodated for and provides more opportunities for cloud-free, non-missing and atmospherically uncontaminated surface observations. Second, the adoption of an object based approach, namely the variational region-based geometric active contour method that enables robust segmentation with only a small number of parameters and that requires no training data collection. Third, the use of a watershed algorithm to decompose connected segments belonging to multiple fields into coherent isolated field segments and a geometry based algorithm to detect and associate parts of

  17. Automated extraction and semantic analysis of mutation impacts from the biomedical literature.

    PubMed

    Naderi, Nona; Witte, René

    2012-06-18

    ), the first comprehensive, fully open-source approach to automatically extract impacts and related relevant information from the biomedical literature. We assessed the performance of our work on manually annotated corpora and the results show the reliability of our approach. The representation of the extracted information into a structured format facilitates knowledge management and aids in database curation and correction. Furthermore, access to the analysis results is provided through multiple interfaces, including web services for automated data integration and desktop-based solutions for end user interactions.

  18. Novel extraction strategy of ribosomal RNA and genomic DNA from cheese for PCR-based investigations.

    PubMed

    Bonaïti, Catherine; Parayre, Sandrine; Irlinger, Françoise

    2006-03-15

    Cheese microorganisms, such as bacteria and fungi, constitute a complex ecosystem that plays a central role in cheeses ripening. The molecular study of cheese microbial diversity and activity is essential but the extraction of high quality nucleic acid may be problematic: the cheese samples are characterised by a strong buffering capacity which negatively influenced the yield of the extracted rRNA. The objective of this study is to develop an effective method for the direct and simultaneous isolation of yeast and bacterial ribosomal RNA and genomic DNA from the same cheese samples. DNA isolation was based on a protocol used for nucleic acids isolation from anaerobic digestor, without preliminary washing step with the combined use of the action of chaotropic agent (acid guanidinium thiocyanate), detergents (SDS, N-lauroylsarcosine), chelating agent (EDTA) and a mechanical method (bead beating system). The DNA purification was carried out by two washing steps of phenol-chloroform. RNA was isolated successfully after the second acid extraction step by recovering it from the phenolic phase of the first acid extraction. The novel method yielded pure preparation of undegraded RNA accessible for reverse transcription-PCR. The extraction protocol of genomic DNA and rRNA was applicable to complex ecosystem of different cheese matrices.

  19. Quality Control of RNA Preservation and Extraction from Paraffin-Embedded Tissue: Implications for RT-PCR and Microarray Analysis

    PubMed Central

    Pichler, Martin; Zatloukal, Kurt

    2013-01-01

    Analysis of RNA isolated from fixed and paraffin-embedded tissues is widely used in biomedical research and molecular pathological diagnostics. We have performed a comprehensive and systematic investigation of the impact of factors in the pre-analytical workflow, such as different fixatives, fixation time, RNA extraction method and storage of tissues in paraffin blocks, on several downstream reactions including complementary DNA (cDNA) synthesis, quantitative reverse transcription polymerase chain reaction (qRT-PCR) and microarray hybridization. We compared the effects of routine formalin fixation with the non-crosslinking, alcohol-based Tissue Tek Xpress Molecular Fixative (TTXMF, Sakura Finetek), and cryopreservation as gold standard for molecular analyses. Formalin fixation introduced major changes into microarray gene expression data and led to marked gene-to-gene variations in delta-ct values of qRT-PCR. We found that qRT-PCR efficiency and gene-to-gene variations were mainly attributed to differences in the efficiency of cDNA synthesis as the most sensitive step. These differences could not be reliably detected by quality assessment of total RNA isolated from formalin-fixed tissues by electrophoresis or spectrophotometry. Although RNA from TTXMF fixed samples was as fragmented as RNA from formalin fixed samples, much higher cDNA yield and lower ct-values were obtained in qRT-PCR underlining the negative impact of crosslinking by formalin. In order to better estimate the impact of pre-analytical procedures such as fixation on the reliability of downstream analysis, we applied a qRT-PCR-based assay using amplicons of different length and an assay measuring the efficiency of cDNA generation. Together these two assays allowed better quality assessment of RNA extracted from fixed and paraffin-embedded tissues and should be used to supplement quality scores derived from automated electrophoresis. A better standardization of the pre-analytical workflow, application

  20. Quality control of RNA preservation and extraction from paraffin-embedded tissue: implications for RT-PCR and microarray analysis.

    PubMed

    Kashofer, Karl; Viertler, Christian; Pichler, Martin; Zatloukal, Kurt

    2013-01-01

    Analysis of RNA isolated from fixed and paraffin-embedded tissues is widely used in biomedical research and molecular pathological diagnostics. We have performed a comprehensive and systematic investigation of the impact of factors in the pre-analytical workflow, such as different fixatives, fixation time, RNA extraction method and storage of tissues in paraffin blocks, on several downstream reactions including complementary DNA (cDNA) synthesis, quantitative reverse transcription polymerase chain reaction (qRT-PCR) and microarray hybridization. We compared the effects of routine formalin fixation with the non-crosslinking, alcohol-based Tissue Tek Xpress Molecular Fixative (TTXMF, Sakura Finetek), and cryopreservation as gold standard for molecular analyses. Formalin fixation introduced major changes into microarray gene expression data and led to marked gene-to-gene variations in delta-ct values of qRT-PCR. We found that qRT-PCR efficiency and gene-to-gene variations were mainly attributed to differences in the efficiency of cDNA synthesis as the most sensitive step. These differences could not be reliably detected by quality assessment of total RNA isolated from formalin-fixed tissues by electrophoresis or spectrophotometry. Although RNA from TTXMF fixed samples was as fragmented as RNA from formalin fixed samples, much higher cDNA yield and lower ct-values were obtained in qRT-PCR underlining the negative impact of crosslinking by formalin. In order to better estimate the impact of pre-analytical procedures such as fixation on the reliability of downstream analysis, we applied a qRT-PCR-based assay using amplicons of different length and an assay measuring the efficiency of cDNA generation. Together these two assays allowed better quality assessment of RNA extracted from fixed and paraffin-embedded tissues and should be used to supplement quality scores derived from automated electrophoresis. A better standardization of the pre-analytical workflow, application

  1. 16S rRNA Gene Sequence Analysis of Drinking Water Using RNA and DNA Extracts as Targets for Clone Library Development

    EPA Science Inventory

    The bacterial composition of chlorinated drinking water was analyzed using 16S rRNA gene clone libraries derived from DNA extracts of 12 samples and compared to clone libraries previously generated using RNA extracts from the same samples. Phylogenetic analysis of 761 DNA-based ...

  2. 16S rRNA Gene Sequence Analysis of Drinking Water Using RNA and DNA Extracts as Targets for Clone Library Development

    EPA Science Inventory

    The bacterial composition of chlorinated drinking water was analyzed using 16S rRNA gene clone libraries derived from DNA extracts of 12 samples and compared to clone libraries previously generated using RNA extracts from the same samples. Phylogenetic analysis of 761 DNA-based ...

  3. High-quality RNA extraction from the sea urchin Paracentrotus lividus embryos

    PubMed Central

    Ruocco, Nadia; Costantini, Susan; Zupo, Valerio; Romano, Giovanna; Ianora, Adrianna; Fontana, Angelo; Costantini, Maria

    2017-01-01

    The sea urchin Paracentrotus lividus (Lamarck, 1816) is a keystone herbivore in the Mediterranean Sea due to its ability to transform macroalgal-dominated communities into barren areas characterized by increased cover of bare substrates and encrusting coralline algae, reduced biodiversity and altered ecosystem functions. P. lividus is also an excellent animal model for toxicology, physiology and biology investigations having been used for more than a century as a model for embryological studies with synchronously developing embryos which are easy to manipulate and analyze for morphological aberrations. Despite its importance for the scientific community, the complete genome is still not fully annotated. To date, only a few molecular tools are available and a few Next Generation Sequencing (NGS) studies have been performed. Here we aimed at setting-up an RNA extraction method to obtain high quality and sufficient quantity of RNA for NGS from P. lividus embryos at the pluteus stage. We compared five different RNA extraction protocols from four different pools of plutei (500, 1000, 2500 and 5000 embryos): TRIzol®, and four widely-used Silica Membrane kits, GenElute™ Mammalian Total RNA Miniprep Kit, RNAqueous® Micro Kit, RNeasy® Micro Kit and Aurum™ Total RNA Mini Kit. The quantity of RNA isolated was evaluated using NanoDrop. The quality, considering the purity, was measured as A260/A280 and A260/230 ratios. The integrity was measured by RNA Integrity Number (RIN). Our results demonstrated that the most efficient procedures were GenElute, RNeasy and Aurum, producing a sufficient quantity of RNA for NGS. The Bioanalyzer profiles and RIN values revealed that the most efficient methods guaranteeing for RNA integrity were RNeasy and Aurum combined with an initial preservation in RNAlater. This research represents the first attempt to standardize a method for high-quality RNA extraction from sea urchin embryos at the pluteus stage, providing a new resource for this

  4. High-quality RNA extraction from the sea urchin Paracentrotus lividus embryos.

    PubMed

    Ruocco, Nadia; Costantini, Susan; Zupo, Valerio; Romano, Giovanna; Ianora, Adrianna; Fontana, Angelo; Costantini, Maria

    2017-01-01

    The sea urchin Paracentrotus lividus (Lamarck, 1816) is a keystone herbivore in the Mediterranean Sea due to its ability to transform macroalgal-dominated communities into barren areas characterized by increased cover of bare substrates and encrusting coralline algae, reduced biodiversity and altered ecosystem functions. P. lividus is also an excellent animal model for toxicology, physiology and biology investigations having been used for more than a century as a model for embryological studies with synchronously developing embryos which are easy to manipulate and analyze for morphological aberrations. Despite its importance for the scientific community, the complete genome is still not fully annotated. To date, only a few molecular tools are available and a few Next Generation Sequencing (NGS) studies have been performed. Here we aimed at setting-up an RNA extraction method to obtain high quality and sufficient quantity of RNA for NGS from P. lividus embryos at the pluteus stage. We compared five different RNA extraction protocols from four different pools of plutei (500, 1000, 2500 and 5000 embryos): TRIzol®, and four widely-used Silica Membrane kits, GenElute™ Mammalian Total RNA Miniprep Kit, RNAqueous® Micro Kit, RNeasy® Micro Kit and Aurum™ Total RNA Mini Kit. The quantity of RNA isolated was evaluated using NanoDrop. The quality, considering the purity, was measured as A260/A280 and A260/230 ratios. The integrity was measured by RNA Integrity Number (RIN). Our results demonstrated that the most efficient procedures were GenElute, RNeasy and Aurum, producing a sufficient quantity of RNA for NGS. The Bioanalyzer profiles and RIN values revealed that the most efficient methods guaranteeing for RNA integrity were RNeasy and Aurum combined with an initial preservation in RNAlater. This research represents the first attempt to standardize a method for high-quality RNA extraction from sea urchin embryos at the pluteus stage, providing a new resource for this

  5. A microfluidic approach for high efficiency extraction of low molecular weight RNA.

    PubMed

    Vulto, Paul; Dame, Gregory; Maier, Urban; Makohliso, Solomzi; Podszun, Susann; Zahn, Peter; Urban, Gerald A

    2010-03-07

    The lack of sample pre-treatment concepts that are easily automatable, miniaturized and highly efficient for both small volumes and low target concentrations, is one of the key issues that block the road towards effective miniaturized diagnostic instruments. This paper presents a novel, highly efficient and simple method for low-molecular weight RNA extraction using electricity only. Cells are lysed by thermo-electric lysis and RNA is purified using a gel-electrophoretic purification step. The combination of the two steps in one integrated cartridge reduces the time frame between the two steps, thus protecting RNA from enzymatic degradation. A disposable chip solution is proposed using a novel dry film resist laminate technology that allows cheap, large-scale fabrication. The chip contains crucial microfluidic innovations that allow for a simple user interface, reproducible functioning and precise quantification. Phaseguides are invented that allow controlled spatial injection of gel, injection of sample and recovery of extracted RNA. A precise sample volume can be defined by integrating electrophoretic actuation electrodes in the microfluidic chamber. Electrolytic gas bubbles that are the result of constant-current actuation are driven out from the chip by the novel introduction of capillary bubble-expulsion techniques. The extraction approach and the functionality of the chip are demonstrated for Escherichia coli and Streptococcus thermophilus bacteria. Linear extraction behavior is obtained for transfer-messenger RNA down to one colony-forming unit per microlitre, or five colony-forming units per chip. The latter is an increase in extraction efficiency of a factor of 1000 with respect to the commercial extraction kit Ambion Ribopure. The chip shows particularly good performance for extraction of low-molecular weight RNA, thereby eliminating the need for large ribosomal RNA and DNA removal. RNA can be extracted in less than 11 min, being a speed-up of more than a

  6. The Influence of DNA Extraction Procedure and Primer Set on the Bacterial Community Analysis by Pyrosequencing of Barcoded 16S rRNA Gene Amplicons

    PubMed Central

    Starke, Ingo C.; Vahjen, Wilfried; Pieper, Robert; Zentek, Jürgen

    2014-01-01

    In this study, the effect of different DNA extraction procedures and primer sets on pyrosequencing results regarding the composition of bacterial communities in the ileum of piglets was investigated. Ileal chyme from piglets fed a diet containing different amounts of zinc oxide was used to evaluate a pyrosequencing study with barcoded 16S rRNA PCR products. Two DNA extraction methods (bead beating versus silica gel columns) and two primer sets targeting variable regions of bacterial 16S rRNA genes (8f-534r versus 968f-1401r) were considered. The SEED viewer software of the MG-RAST server was used for automated sequence analysis. A total of 5.2 × 105 sequences were used for analysis after processing for read length (150 bp), minimum sequence occurrence (5), and exclusion of eukaryotic and unclassified/uncultured sequences. DNA extraction procedures and primer sets differed significantly in total sequence yield. The distribution of bacterial order and main bacterial genera was influenced significantly by both parameters. However, this study has shown that the results of pyrosequencing studies using barcoded PCR amplicons of bacterial 16S rRNA genes depend on DNA extraction and primer choice, as well as on the manner of downstream sequence analysis. PMID:25120931

  7. A comparison of methods for forensic DNA extraction: Chelex-100® and the QIAGEN DNA Investigator Kit (manual and automated).

    PubMed

    Phillips, Kirsty; McCallum, Nicola; Welch, Lindsey

    2012-03-01

    Efficient isolation of DNA from a sample is the basis for successful forensic DNA profiling. There are many DNA extraction methods available and they vary in their ability to efficiently extract the DNA; as well as in processing time, operator intervention, contamination risk and ease of use. In recent years, automated robots have been made available which speed up processing time and decrease the amount of operator input. This project was set up to investigate the efficiency of three DNA extraction methods, two manual (Chelex(®)-100 and the QIAGEN DNA Investigator Kit) and one automated (QIAcube), using both buccal cells and blood stains as the DNA source. Extracted DNA was quantified using real-time PCR in order to assess the amount of DNA present in each sample. Selected samples were then amplified using AmpFlSTR SGM Plus amplification kit. The results suggested that there was no statistical difference between results gained for the different methods investigated, but the automated QIAcube robot made sample processing much simpler and quicker without introducing DNA contamination.

  8. A new automated spectral feature extraction method and its application in spectral classification and defective spectra recovery

    NASA Astrophysics Data System (ADS)

    Wang, Ke; Guo, Ping; Luo, A.-Li

    2017-03-01

    Spectral feature extraction is a crucial procedure in automated spectral analysis. This procedure starts from the spectral data and produces informative and non-redundant features, facilitating the subsequent automated processing and analysis with machine-learning and data-mining techniques. In this paper, we present a new automated feature extraction method for astronomical spectra, with application in spectral classification and defective spectra recovery. The basic idea of our approach is to train a deep neural network to extract features of spectra with different levels of abstraction in different layers. The deep neural network is trained with a fast layer-wise learning algorithm in an analytical way without any iterative optimization procedure. We evaluate the performance of the proposed scheme on real-world spectral data. The results demonstrate that our method is superior regarding its comprehensive performance, and the computational cost is significantly lower than that for other methods. The proposed method can be regarded as a new valid alternative general-purpose feature extraction method for various tasks in spectral data analysis.

  9. Improvement on the extraction method of RNA in mites and its quality test.

    PubMed

    Zhao, YaE; Hu, Li; Yang, Yuan Jun; Niu, Dong Ling; Wang, Rui Ling; Li, Wen Hao; Ma, Si Jia; Cheng, Juan

    2016-02-01

    To solve the long-existing difficult problems in extracting RNA and constructing a complementary DNA (cDNA) library for trace mites, we conducted a further comparative experiment among three RNA extraction methods (TRIzol method, Omega method, and Azanno method) based on our previous attempts at the construction of cDNA library of mites, with Psoroptes cuniculi still used as the experimental subject. By subsequently decreasing the number of mites, the least number of mites needed for RNA extraction of each method were found by criteria of completeness, concentration, and purity of the extracted RNA. Specific primers were designed according to the allergen Pso c1, Pso c2, and Actin gene sequences of Psoroptes to test the reliability of cDNA library. The results showed that Azanno method needed only 10 mites with sensitivity 204 times higher than previously used TRIzol method and 20 times higher than Omega method; clear RNA band was detected by agarose gel electrophoresis; and ultraviolet spectrophotometer determination showed that RNA concentration, 260/280, and 260/230 were in the range of 102 to 166 ng/μl, 1.83 to 1.99, and 1.49 to 1.72, respectively. Finally, specific primers detection showed that the amplified sequences had 98.33, 98.19, and 99.52% identities with those of P. cuniculi or Psoroptes ovis in GenBank, respectively, indicating that the cDNA library constructed using 10 mites was successful and it could meet the requirements for molecular biology research. Therefore, we concluded that Azanno method was more effective than TRIzol method and Omega method in RNA extraction and cDNA library construction of trace mites.

  10. A methodology for automated CPA extraction using liver biopsy image analysis and machine learning techniques.

    PubMed

    Tsipouras, Markos G; Giannakeas, Nikolaos; Tzallas, Alexandros T; Tsianou, Zoe E; Manousou, Pinelopi; Hall, Andrew; Tsoulos, Ioannis; Tsianos, Epameinondas

    2017-03-01

    Collagen proportional area (CPA) extraction in liver biopsy images provides the degree of fibrosis expansion in liver tissue, which is the most characteristic histological alteration in hepatitis C virus (HCV). Assessment of the fibrotic tissue is currently based on semiquantitative staging scores such as Ishak and Metavir. Since its introduction as a fibrotic tissue assessment technique, CPA calculation based on image analysis techniques has proven to be more accurate than semiquantitative scores. However, CPA has yet to reach everyday clinical practice, since the lack of standardized and robust methods for computerized image analysis for CPA assessment have proven to be a major limitation. The current work introduces a three-stage fully automated methodology for CPA extraction based on machine learning techniques. Specifically, clustering algorithms have been employed for background-tissue separation, as well as for fibrosis detection in liver tissue regions, in the first and the third stage of the methodology, respectively. Due to the existence of several types of tissue regions in the image (such as blood clots, muscle tissue, structural collagen, etc.), classification algorithms have been employed to identify liver tissue regions and exclude all other non-liver tissue regions from CPA computation. For the evaluation of the methodology, 79 liver biopsy images have been employed, obtaining 1.31% mean absolute CPA error, with 0.923 concordance correlation coefficient. The proposed methodology is designed to (i) avoid manual threshold-based and region selection processes, widely used in similar approaches presented in the literature, and (ii) minimize CPA calculation time. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  11. Reliability and performance of commercial RNA and DNA extraction kits for FFPE tissue cores

    PubMed Central

    Guérard, Karl-Philippe; Bartlett, John M. S.; Lapointe, Jacques; Berman, David M.; Park, Paul C.

    2017-01-01

    Cancer biomarker studies often require nucleic acid extraction from limited amounts of formalin-fixed, paraffin-embedded (FFPE) tissues, such as histologic sections or needle cores. A major challenge is low quantity and quality of extracted nucleic acids, which can limit our ability to perform genetic analyses, and have a significant influence on overall study design. This study was aimed at identifying the most reliable and reproducible method of obtaining sufficient high-quality nucleic acids from FFPE tissues. We compared the yield and quality of nucleic acids from 0.6-mm FFPE prostate tissue cores across 16 DNA and RNA extraction protocols, using 14 commercially available kits. Nucleic acid yield was determined by fluorometry, and quality was determined by spectrophotometry. All protocols yielded nucleic acids in quantities that are compatible with downstream molecular applications. However, the protocols varied widely in the quality of the extracted RNA and DNA. Four RNA and five DNA extraction protocols, including protocols from two kits for dual-extraction of RNA and DNA from the same tissue source, were prioritized for further quality assessment based on the yield and purity of their products. Specifically, their compatibility with downstream reactions was assessed using both NanoString nCounter gene expression assays and reverse-transcriptase real-time PCR for RNA, and methylation-specific PCR assays for DNA. The kit deemed most suitable for FFPE tissue was the AllPrep kit by Qiagen because of its yield, quality, and ability to purify both RNA and DNA from the same sample, which would be advantageous in biomarker studies. PMID:28640876

  12. Reliability and performance of commercial RNA and DNA extraction kits for FFPE tissue cores.

    PubMed

    Patel, Palak G; Selvarajah, Shamini; Guérard, Karl-Philippe; Bartlett, John M S; Lapointe, Jacques; Berman, David M; Okello, John B A; Park, Paul C

    2017-01-01

    Cancer biomarker studies often require nucleic acid extraction from limited amounts of formalin-fixed, paraffin-embedded (FFPE) tissues, such as histologic sections or needle cores. A major challenge is low quantity and quality of extracted nucleic acids, which can limit our ability to perform genetic analyses, and have a significant influence on overall study design. This study was aimed at identifying the most reliable and reproducible method of obtaining sufficient high-quality nucleic acids from FFPE tissues. We compared the yield and quality of nucleic acids from 0.6-mm FFPE prostate tissue cores across 16 DNA and RNA extraction protocols, using 14 commercially available kits. Nucleic acid yield was determined by fluorometry, and quality was determined by spectrophotometry. All protocols yielded nucleic acids in quantities that are compatible with downstream molecular applications. However, the protocols varied widely in the quality of the extracted RNA and DNA. Four RNA and five DNA extraction protocols, including protocols from two kits for dual-extraction of RNA and DNA from the same tissue source, were prioritized for further quality assessment based on the yield and purity of their products. Specifically, their compatibility with downstream reactions was assessed using both NanoString nCounter gene expression assays and reverse-transcriptase real-time PCR for RNA, and methylation-specific PCR assays for DNA. The kit deemed most suitable for FFPE tissue was the AllPrep kit by Qiagen because of its yield, quality, and ability to purify both RNA and DNA from the same sample, which would be advantageous in biomarker studies.

  13. A multi-atlas based method for automated anatomical rat brain MRI segmentation and extraction of PET activity.

    PubMed

    Lancelot, Sophie; Roche, Roxane; Slimen, Afifa; Bouillot, Caroline; Levigoureux, Elise; Langlois, Jean-Baptiste; Zimmer, Luc; Costes, Nicolas

    2014-01-01

    Preclinical in vivo imaging requires precise and reproducible delineation of brain structures. Manual segmentation is time consuming and operator dependent. Automated segmentation as usually performed via single atlas registration fails to account for anatomo-physiological variability. We present, evaluate, and make available a multi-atlas approach for automatically segmenting rat brain MRI and extracting PET activies. High-resolution 7T 2DT2 MR images of 12 Sprague-Dawley rat brains were manually segmented into 27-VOI label volumes using detailed protocols. Automated methods were developed with 7/12 atlas datasets, i.e. the MRIs and their associated label volumes. MRIs were registered to a common space, where an MRI template and a maximum probability atlas were created. Three automated methods were tested: 1/registering individual MRIs to the template, and using a single atlas (SA), 2/using the maximum probability atlas (MP), and 3/registering the MRIs from the multi-atlas dataset to an individual MRI, propagating the label volumes and fusing them in individual MRI space (propagation & fusion, PF). Evaluation was performed on the five remaining rats which additionally underwent [18F]FDG PET. Automated and manual segmentations were compared for morphometric performance (assessed by comparing volume bias and Dice overlap index) and functional performance (evaluated by comparing extracted PET measures). Only the SA method showed volume bias. Dice indices were significantly different between methods (PF>MP>SA). PET regional measures were more accurate with multi-atlas methods than with SA method. Multi-atlas methods outperform SA for automated anatomical brain segmentation and PET measure's extraction. They perform comparably to manual segmentation for FDG-PET quantification. Multi-atlas methods are suitable for rapid reproducible VOI analyses.

  14. Methylation of tRNA by normal mouse liver and Erhlich ascites cell extracts

    SciTech Connect

    Smith, S.D.; Sitz, T.O.

    1986-05-01

    When Erhlich ascites tumor and normal mouse liver extracts were incubated with (/sup 3/H-CH/sub 3/)-S-adenosylmethionine and E. coli tRNA, the tumor extracts had a ten-fold higher methyltransferase activity than mouse liver extracts. However, only a two-fold higher methyltransferase activity was observed when methyl deficient mouse liver tRNA was used as a substrate. DEAE-Sephadex chromatography of alkaline hydrolysates of methyl deficient mouse liver tRNA (tRNA isolated from mice treated with ethionine) showed 70% of the /sup 3/H-methyl groups in the mononucleotide peak and 30% in alkaline stable di-, tri- and oligonucleotide peaks demonstrating methylation of both the base and ribose moieties. Perchloric acid digest of material isolated from the di-, tri- and oligonucleotide peaks confirm that the /sup 3/H-methyl groups are on the ribose moieties. In hydrolysates of E. coli tRNA /sup 3/H-methyl groups appeared in only the mononucleotide peak, i.e. only base methylation. These data, from Erhlich ascites cells, suggest that ribose methyltransferases may have greater RNA substrate specificity than the base methyltransferases and may act processively to produce alkaline stable oligonucleotides.

  15. A method for extracting and characterizing RNA from urine: For downstream PCR and RNAseq analysis.

    PubMed

    Zhou, Kun; Spillman, Monique A; Behbakht, Kian; Komatsu, Julia M; Abrahante, Juan E; Hicks, Douglas; Schotl, Brent; Odean, Evan; Jones, Kenneth L; Graner, Michael W; Bemis, Lynne T

    2017-08-10

    Readily accessible samples such as urine or blood are seemingly ideal for differentiating and stratifying patients; however, it has proven a daunting task to identify reliable biomarkers in such samples. Noncoding RNA holds great promise as a source of biomarkers distinguishing physiologic wellbeing or illness. Current methods to isolate and characterize RNA molecules in urine are limited. In this proof of concept study, we present a method to extract and identify small noncoding RNAs in urine. Initially, quantitative reverse transcription PCR was applied to confirm the presence of microRNAs in total RNA extracted from urine. Once the presence of micro RNA in urine was confirmed, we developed a method to scale up RNA extraction to provide adequate amounts of RNA for next generation sequence analysis. The method described in this study is applicable to detecting a broad range of small noncoding RNAs in urine; thus, they have wide applicability for health and disease analyses. Copyright © 2017 Elsevier Inc. All rights reserved.

  16. Sensitivity testing of trypanosome detection by PCR from whole blood samples using manual and automated DNA extraction methods.

    PubMed

    Dunlop, J; Thompson, C K; Godfrey, S S; Thompson, R C A

    2014-11-01

    Automated extraction of DNA for testing of laboratory samples is an attractive alternative to labour-intensive manual methods when higher throughput is required. However, it is important to maintain the maximum detection sensitivity possible to reduce the occurrence of type II errors (false negatives; failure to detect the target when it is present), especially in the biomedical field, where PCR is used for diagnosis. We used blood infected with known concentrations of Trypanosoma copemani to test the impact of analysis techniques on trypanosome detection sensitivity by PCR. We compared combinations of a manual and an automated DNA extraction method and two different PCR primer sets to investigate the impact of each on detection levels. Both extraction techniques and specificity of primer sets had a significant impact on detection sensitivity. Samples extracted using the same DNA extraction technique performed substantially differently for each of the separate primer sets. Type I errors (false positives; detection of the target when it is not present), produced by contaminants, were avoided with both extraction methods. This study highlights the importance of testing laboratory techniques with known samples to optimise accuracy of test results.

  17. The BUME method: a novel automated chloroform-free 96-well total lipid extraction method for blood plasma[S

    PubMed Central

    Löfgren, Lars; Ståhlman, Marcus; Forsberg, Gun-Britt; Saarinen, Sinikka; Nilsson, Ralf; Hansson, Göran I.

    2012-01-01

    Lipid extraction from biological samples is a critical and often tedious preanalytical step in lipid research. Primarily on the basis of automation criteria, we have developed the BUME method, a novel chloroform-free total lipid extraction method for blood plasma compatible with standard 96-well robots. In only 60 min, 96 samples can be automatically extracted with lipid profiles of commonly analyzed lipid classes almost identically and with absolute recoveries similar or better to what is obtained using the chloroform-based reference method. Lipid recoveries were linear from 10–100 µl plasma for all investigated lipids using the developed extraction protocol. The BUME protocol includes an initial one-phase extraction of plasma into 300 µl butanol:methanol (BUME) mixture (3:1) followed by two-phase extraction into 300 µl heptane:ethyl acetate (3:1) using 300 µl 1% acetic acid as buffer. The lipids investigated included the most abundant plasma lipid classes (e.g., cholesterol ester, free cholesterol, triacylglycerol, phosphatidylcholine, and sphingomyelin) as well as less abundant but biologically important lipid classes, including ceramide, diacylglycerol, and lyso-phospholipids. This novel method has been successfully implemented in our laboratory and is now used daily. We conclude that the fully automated, high-throughput BUME method can replace chloroform-based methods, saving both human and environmental resources. PMID:22645248

  18. From clinical sample to complete genome: Comparing methods for the extraction of HIV-1 RNA for high-throughput deep sequencing.

    PubMed

    Cornelissen, Marion; Gall, Astrid; Vink, Monique; Zorgdrager, Fokla; Binter, Špela; Edwards, Stephanie; Jurriaans, Suzanne; Bakker, Margreet; Ong, Swee Hoe; Gras, Luuk; van Sighem, Ard; Bezemer, Daniela; de Wolf, Frank; Reiss, Peter; Kellam, Paul; Berkhout, Ben; Fraser, Christophe; van der Kuyl, Antoinette C

    2016-08-04

    The BEEHIVE (Bridging the Evolution and Epidemiology of HIV in Europe) project aims to analyse nearly-complete viral genomes from >3000 HIV-1 infected Europeans using high-throughput deep sequencing techniques to investigate the virus genetic contribution to virulence. Following the development of a computational pipeline, including a new de novo assembler for RNA virus genomes, to generate larger contiguous sequences (contigs) from the abundance of short sequence reads that characterise the data, another area that determines genome sequencing success is the quality and quantity of the input RNA. A pilot experiment with 125 patient plasma samples was performed to investigate the optimal method for isolation of HIV-1 viral RNA for long amplicon genome sequencing. Manual isolation with the QIAamp Viral RNA Mini Kit (Qiagen) was superior over robotically extracted RNA using either the QIAcube robotic system, the mSample Preparation Systems RNA kit with automated extraction by the m2000sp system (Abbott Molecular), or the MagNA Pure 96 System in combination with the MagNA Pure 96 Instrument (Roche Diagnostics). We scored amplification of a set of four HIV-1 amplicons of ∼1.9, 3.6, 3.0 and 3.5kb, and subsequent recovery of near-complete viral genomes. Subsequently, 616 BEEHIVE patient samples were analysed to determine factors that influence successful amplification of the genome in four overlapping amplicons using the QIAamp Viral RNA Kit for viral RNA isolation. Both low plasma viral load and high sample age (stored before 1999) negatively influenced the amplification of viral amplicons >3kb. A plasma viral load of >100,000 copies/ml resulted in successful amplification of all four amplicons for 86% of the samples, this value dropped to only 46% for samples with viral loads of <20,000 copies/ml.

  19. How Severely Is DNA Quantification Hampered by RNA Co-extraction?

    PubMed

    Sanchez, Ignacio; Remm, Matthieu; Frasquilho, Sonia; Betsou, Fay; Mathieson, William

    2015-10-01

    The optional RNase digest that is part of many DNA extraction protocols is often omitted, either because RNase is not provided in the kit or because users do not want to risk contaminating their laboratory. Consequently, co-eluting RNA can become a "contaminant" of unknown magnitude in a DNA extraction. We extracted DNA from liver, lung, kidney, and heart tissues and established that 28-52% of the "DNA" as assessed by spectrophotometry is actually RNA (depending on tissue type). Including an RNase digest in the extraction protocol reduced 260:280 purity ratios. Co-eluting RNA drives an overestimation of DNA yield when quantification is carried out using OD 260 nm spectrophotometry, or becomes an unquantified contaminant when spectrofluorometry is used for DNA quantification. This situation is potentially incompatible with the best practice guidelines for biobanks issued by organizations such as the International Society for Biological and Environmental Repositories, which state that biospecimens should be accurately characterized in terms of their identity, purity, concentration, and integrity. Consequently, we conclude that an RNase digest must be included in DNA extractions if pure DNA is required. We also discuss the implications of unquantified RNA contamination in DNA samples in the context of laboratory accreditation schemes.

  20. In vitro RNA editing-like activity in a mitochondrial extract from Leishmania tarentolae.

    PubMed Central

    Frech, G C; Bakalara, N; Simpson, L; Simpson, A M

    1995-01-01

    A mitochondrial extract from Leishmania tarentolae directs the incorporation of uridylate (U) residues within the pre-edited domain of synthetic cytochrome b (CYb) and NADH dehydrogenase subunit 7 mRNA. This has several characteristics of an in vitro RNA editing activity, but no direct evidence for involvement of guide RNAs was obtained. Inhibition by micrococcal nuclease suggests a requirement for some type of endogenous RNA. The limitation of internal U-incorporation to the pre-edited region in the CYb mRNA and the inhibition by deletion or substitution of both mRNA anchor sequences for CYb gRNA-I and -II could be consistent either with a gRNA-mediated process or a secondary structure-mediated process. A low level of incorporation of [alpha-32P]CTP occurs at the same sites as UTP. Internal U-incorporation activity is selectively inhibited by heterologous RNAs, suggesting an involvement of low affinity RNA-binding proteins which can be competed by the added RNA. Images PMID:7828590

  1. Rapid and Semi-Automated Extraction of Neuronal Cell Bodies and Nuclei from Electron Microscopy Image Stacks

    PubMed Central

    Holcomb, Paul S.; Morehead, Michael; Doretto, Gianfranco; Chen, Peter; Berg, Stuart; Plaza, Stephen; Spirou, George

    2016-01-01

    Connectomics—the study of how neurons wire together in the brain—is at the forefront of modern neuroscience research. However, many connectomics studies are limited by the time and precision needed to correctly segment large volumes of electron microscopy (EM) image data. We present here a semi-automated segmentation pipeline using freely available software that can significantly decrease segmentation time for extracting both nuclei and cell bodies from EM image volumes. PMID:27259933

  2. Rapid and Semi-automated Extraction of Neuronal Cell Bodies and Nuclei from Electron Microscopy Image Stacks.

    PubMed

    Holcomb, Paul S; Morehead, Michael; Doretto, Gianfranco; Chen, Peter; Berg, Stuart; Plaza, Stephen; Spirou, George

    2016-01-01

    Connectomics-the study of how neurons wire together in the brain-is at the forefront of modern neuroscience research. However, many connectomics studies are limited by the time and precision needed to correctly segment large volumes of electron microscopy (EM) image data. We present here a semi-automated segmentation pipeline using freely available software that can significantly decrease segmentation time for extracting both nuclei and cell bodies from EM image volumes.

  3. Extraction of total RNA from leaves of Eucalyptus and other woody and herbaceous plants using sodium isoascorbate.

    PubMed

    Suzuki, Y; Hibino, T; Kawazu, T; Wada, T; Kihara, T; Koyama, H

    2003-05-01

    Rapid extraction of total RNA from Eucalyptus leaves is difficult due to the high content of polyphenolics and polysaccharides. A rapid and simple method was developed by using an extraction buffer containing sodium isoascorbate at a concentration of 500 mM. This method consisted of one or two chloroform extractions, one acid guanidium-phenol-chloroform extraction, and isopropanol precipitation alone. The yields of the RNA fractions were 246-1750 micrograms/g fresh weight when leaves of Eucalyptus, five other woody plants, and four herbaceous plants were used as samples. The contamination of the RNA fractions by proteins and polysaccharides was very limited as judged spectrophotometrically. When the RNA fractions were subjected to agarose gel electrophoresis, intact rRNA bands were detected. The RNA fractions could be used for RT-PCR. These results indicate that our new method achieves a simple and rapid preparation of high-quality RNA from leaves of Eucalyptus and other plant species.

  4. Development of an RNA extraction protocol for detection of waterborne viruses by reverse transcriptase quantitative PCR (RT-qPCR).

    PubMed

    Jothikumar, N; Sobsey, M D; Cromeans, T L

    2010-10-01

    RNA extraction from environmental samples yields frequently an RNA preparation containing inhibitors of molecular reactions. Commercial RNA extraction kits commonly permit extraction of only 0.1-0.2 ml sample volume. An RNA extraction buffer (RNAX buffer) was formulated for the extraction of viral RNA from 4.0 ml using a silica column based protocol. To evaluate the RNAX buffer based protocol, we used hepatitis A virus (HAV) and coxsackievirus B3 (CVB3) to monitor the RNA extraction efficiency from environmental samples. For evaluation of viral RNA recovery from water concentrates which were prepared from river and pond water by PEG concentration, serial ten fold dilutions of two waterborne viruses were added to the water concentrates for evaluation by quantitative detection. Quantitative recovery of HAV and CVB3 was determined by reverse transcriptase quantitative real-time PCR (RT-qPCR). The extracted RNA was compatible with RT-qPCR and sensitivity of detection of 0.8PFU per reaction was found with RNAX buffer and the developed protocol. This level of sensitivity was obtained using viral RNA extracted from 4.0 ml of an inoculated water sample concentrate. The RNAX buffer developed in this study could be applicable to the detection of other pathogens in water and food.

  5. Comparison of RNA Extraction Methods for Molecular Analysis of Oral Cytology

    PubMed Central

    Pérez-Sayáns, Mario; Padín-Iruegas, Maria-Elena; Reboiras-López, Maria Dolores; Suarez-Peńaranda, José Manuel; López-López, Rafael; Carta, Celina Faig Lima; Issa, Jaqueline Scholz; García-García, Abel; Almeida, Janete Dias

    2016-01-01

    Objective of work The aim of this study was to compare three methods of RNA extraction for molecular analysis of oral cytology to establish the best technique, considering its concentration and purity for molecular tests of oral lesions such as real-time reverse transcriptase reaction. Material and methods The sample included exfoliative cytology from the oral cavity mucosa of patients with no visible clinical changes, using Orcellex Rovers Brush®. The extraction of total RNA was performed using the following three techniques: 30 samples were extracted by Trizol® technique, 30 by the Direct-zolTM RNA Miniprep system and 30 by the RNeasy mini Kit. The absorbance was measured by spectrophotometer to estimate the purity. The estimated RNA concentration was obtained by multiplying the value of A260 (ng/mL) by 40. Statistical analysis of the obtained data was performed using GraphPad Prism 5.03 software with Student t, analysis of variance and Bonferroni tests, considering p ≤0.05. Results Trizol® group revealed higher average concentration, followed by Direct-zolTM and Rneasy group. It was observed that the RNA Direct-zolTM group had the highest purity, followed by RNeasy and Trizol® groups, allowing for the two ratios. Conclusion Considering all aspects, concentration, purity and time spent in the procedures, the Direct-zolTM group showed the best results. PMID:27789907

  6. High quality RNA extraction from Maqui berry for its application in next-generation sequencing.

    PubMed

    Sánchez, Carolina; Villacreses, Javier; Blanc, Noelle; Espinoza, Loreto; Martinez, Camila; Pastor, Gabriela; Manque, Patricio; Undurraga, Soledad F; Polanco, Victor

    2016-01-01

    Maqui berry (Aristotelia chilensis) is a native Chilean species that produces berries that are exceptionally rich in anthocyanins and natural antioxidants. These natural compounds provide an array of health benefits for humans, making them very desirable in a fruit. At the same time, these substances also interfere with nucleic acid preparations, making RNA extraction from Maqui berry a major challenge. Our group established a method for RNA extraction of Maqui berry with a high quality RNA (good purity, good integrity and higher yield). This procedure is based on the adapted CTAB method using high concentrations of PVP (4 %) and β-mercaptoethanol (4 %) and spermidine in the extraction buffer. These reagents help to remove contaminants such as polysaccharides, proteins, phenols and also prevent the oxidation of phenolic compounds. The high quality of RNA isolated through this method allowed its uses with success in molecular applications for this endemic Chilean fruit, such as differential expression analysis of RNA-Seq data using next generation sequencing (NGS). Furthermore, we consider that our method could potentially be used for other plant species with extremely high levels of antioxidants and anthocyanins.

  7. An Optimized Method for Extracting Bacterial RNA from Mouse Skin Tissue Colonized by Mycobacterium ulcerans.

    PubMed

    Robbe-Saule, Marie; Babonneau, Jérémie; Sismeiro, Odile; Marsollier, Laurent; Marion, Estelle

    2017-01-01

    Bacterial transcriptome analyses during host colonization are essential to decipher the complexity of the relationship between the bacterium and its host. RNA sequencing (RNA-seq) is a promising approach providing valuable information about bacterial adaptation, the host response and, in some cases, mutual tolerance underlying crosstalk, as recently observed in the context of Mycobacterium ulcerans infection. Buruli ulcer is caused by M. ulcerans. This neglected disease is the third most common mycobacterial disease worldwide. Without treatment, M. ulcerans provokes massive skin ulcers. A healing process may be observed in 5% of Buruli ulcer patients several months after the initiation of disease. This spontaneous healing process suggests that some hosts can counteract the development of the lesions caused by M. ulcerans. Deciphering the mechanisms involved in this process should open up new treatment possibilities. To this end, we recently developed the first mouse model for studies of the spontaneous healing process. We have shown that the healing process is based on mutual tolerance between the bacterium and its host. In this context, RNA-seq seems to be the most appropriate method for deciphering bacterial adaptation. However, due to the low bacterial load in host tissues, the isolation of mycobacterial RNA from skin tissue for RNA-seq analysis remains challenging. We developed a method for extracting and purifying mycobacterial RNA whilst minimizing the amount of host RNA in the sample. This approach was based on the extraction of bacterial RNA by a differential lysis method. The challenge in the development of this method was the choice of a lysis system favoring the removal of host RNA without damage to the bacterial cells. We made use of the thick, resistant cell wall of M. ulcerans to achieve this end.

  8. An Optimized Method for Extracting Bacterial RNA from Mouse Skin Tissue Colonized by Mycobacterium ulcerans

    PubMed Central

    Robbe-Saule, Marie; Babonneau, Jérémie; Sismeiro, Odile; Marsollier, Laurent; Marion, Estelle

    2017-01-01

    Bacterial transcriptome analyses during host colonization are essential to decipher the complexity of the relationship between the bacterium and its host. RNA sequencing (RNA-seq) is a promising approach providing valuable information about bacterial adaptation, the host response and, in some cases, mutual tolerance underlying crosstalk, as recently observed in the context of Mycobacterium ulcerans infection. Buruli ulcer is caused by M. ulcerans. This neglected disease is the third most common mycobacterial disease worldwide. Without treatment, M. ulcerans provokes massive skin ulcers. A healing process may be observed in 5% of Buruli ulcer patients several months after the initiation of disease. This spontaneous healing process suggests that some hosts can counteract the development of the lesions caused by M. ulcerans. Deciphering the mechanisms involved in this process should open up new treatment possibilities. To this end, we recently developed the first mouse model for studies of the spontaneous healing process. We have shown that the healing process is based on mutual tolerance between the bacterium and its host. In this context, RNA-seq seems to be the most appropriate method for deciphering bacterial adaptation. However, due to the low bacterial load in host tissues, the isolation of mycobacterial RNA from skin tissue for RNA-seq analysis remains challenging. We developed a method for extracting and purifying mycobacterial RNA whilst minimizing the amount of host RNA in the sample. This approach was based on the extraction of bacterial RNA by a differential lysis method. The challenge in the development of this method was the choice of a lysis system favoring the removal of host RNA without damage to the bacterial cells. We made use of the thick, resistant cell wall of M. ulcerans to achieve this end. PMID:28392785

  9. A simple rapid process for semi-automated brain extraction from magnetic resonance images of the whole mouse head.

    PubMed

    Delora, Adam; Gonzales, Aaron; Medina, Christopher S; Mitchell, Adam; Mohed, Abdul Faheem; Jacobs, Russell E; Bearer, Elaine L

    2016-01-15

    Magnetic resonance imaging (MRI) is a well-developed technique in neuroscience. Limitations in applying MRI to rodent models of neuropsychiatric disorders include the large number of animals required to achieve statistical significance, and the paucity of automation tools for the critical early step in processing, brain extraction, which prepares brain images for alignment and voxel-wise statistics. This novel timesaving automation of template-based brain extraction ("skull-stripping") is capable of quickly and reliably extracting the brain from large numbers of whole head images in a single step. The method is simple to install and requires minimal user interaction. This method is equally applicable to different types of MR images. Results were evaluated with Dice and Jacquard similarity indices and compared in 3D surface projections with other stripping approaches. Statistical comparisons demonstrate that individual variation of brain volumes are preserved. A downloadable software package not otherwise available for extraction of brains from whole head images is included here. This software tool increases speed, can be used with an atlas or a template from within the dataset, and produces masks that need little further refinement. Our new automation can be applied to any MR dataset, since the starting point is a template mask generated specifically for that dataset. The method reliably and rapidly extracts brain images from whole head images, rendering them useable for subsequent analytical processing. This software tool will accelerate the exploitation of mouse models for the investigation of human brain disorders by MRI. Copyright © 2015 Elsevier B.V. All rights reserved.

  10. Automated Agricultural Field Extraction from Multi-temporal Web Enabled Landsat Data

    NASA Astrophysics Data System (ADS)

    Yan, L.; Roy, D. P.

    2012-12-01

    Agriculture has caused significant anthropogenic surface change. In many regions agricultural field sizes may be increasing to maximize yields and reduce costs resulting in decreased landscape spatial complexity and increased homogenization of land uses with potential for significant biogeochemical and ecological effects. To date, studies of the incidence, drivers and impacts of changing field sizes have not been undertaken over large areas because of computational constraints and because consistently processed appropriate resolution data have not been available or affordable. The Landsat series of satellites provides near-global coverage, long term, and appropriate spatial resolution (30m) satellite data to document changing field sizes. The recent free availability of all the Landsat data in the U.S. Landsat archive now provides the opportunity to study field size changes in a global and consistent way. Commercial software can be used to extract fields from Landsat data but are inappropriate for large area application because they require considerable human interaction. This paper presents research to develop and validate an automated computational Geographic Object Based Image Analysis methodology to extract agricultural fields and derive field sizes from Web Enabled Landsat Data (WELD) (http://weld.cr.usgs.gov/). WELD weekly products (30m reflectance and brightness temperature) are classified into Satellite Image Automatic Mapper™ (SIAM™) spectral categories and an edge intensity map and a map of the probability of each pixel being agricultural are derived from five years of 52 weeks of WELD and corresponding SIAM™ data. These data are fused to derive candidate agriculture field segments using a variational region-based geometric active contour model. Geometry-based algorithms are used to decompose connected segments belonging to multiple fields into coherent isolated field objects with a divide and conquer strategy to detect and merge partial circle

  11. Simultaneously extracting DNA, RNA, and protein using kits: is sample quantity or quality prejudiced?

    PubMed

    Mathieson, William; Thomas, Gerry A

    2013-02-01

    Interdisciplinary "omics" research and the stringent quality requirements of array-based technologies require the simultaneous yet efficient extraction of DNA, RNA, and protein from the same tissue block. However, the few commercially available simultaneous extraction kits have not been evaluated. We compare the TriplePrep (GE Healthcare) and AllPrep (Qiagen) kits using good, intermediate, and poor quality tissue with specialist single-extract methods: Puregene (DNA), RNeasy (RNA), and homogenizations into buffer (protein). The following parameters were evaluated: DNA-yield (total DNA and double-stranded), purity (260:280 and 260:230), and integrity (gel electrophoresis); RNA-yield, purity, and integrity (RNA integrity numbers [RINs] and quantitative reverse transcription polymerase chain reaction [Q-RT-PCR]); protein-yield and quality (two-dimensional difference gel electrophoresis [2D-DIGE]). Puregene DNA yields were 183% and 506% those of TriplePrep and AllPrep, respectively. For RNA, AllPrep and RNeasy were indistinguishable, but their yields were 412% to 588% those of TriplePrep (depending on block condition) and their between-sample variability was better. TriplePrep protein yields were 57% those of the control, and 6.9% of the gel spots were more than 2-fold altered. However, AllPrep yields were 20% of the control, with 11% of the gel spots being more than 2-fold altered. Therefore, TriplePrep outperformed AllPrep in DNA and protein extractions, the reverse was true for RNA, but neither kit achieved optimal efficiency because both yield and quality were compromised. Copyright © 2012 Elsevier Inc. All rights reserved.

  12. The use of carrier RNA to enhance DNA extraction from microfluidic-based silica monoliths.

    PubMed

    Shaw, Kirsty J; Thain, Lauren; Docker, Peter T; Dyer, Charlotte E; Greenman, John; Greenway, Gillian M; Haswell, Stephen J

    2009-10-12

    DNA extraction was carried out on silica-based monoliths within a microfluidic device. Solid-phase DNA extraction methodology was applied in which the DNA binds to silica in the presence of a chaotropic salt, such as guanidine hydrochloride, and is eluted in a low ionic strength solution, such as water. The addition of poly-A carrier RNA to the chaotropic salt solution resulted in a marked increase in the effective amount of DNA that could be recovered (25ng) compared to the absence of RNA (5ng) using the silica-based monolith. These findings confirm that techniques utilising nucleic acid carrier molecules can enhance DNA extraction methodologies in microfluidic applications.

  13. Development of a low resource RNA extraction cassette based on surface tension valves

    PubMed Central

    Bordelon, Hali; Adams, Nicholas M.; Klemm, Amy S.; Russ, Patricia K.; Williams, John V.; Talbot, H. Keipp; Wright, David W.; Haselton, Frederick R.

    2011-01-01

    Nucleic acid-based diagnostics are highly sensitive and specific, but are easily disrupted by the presence of interferents in biological samples. In a laboratory or hospital setting, the influence of these interferents can be minimized using an RNA or DNA extraction procedure prior to analysis. However, in low resource settings, limited access to specialized instrumentation and trained personnel presents challenges that impede sample preparation. We have developed a self-contained nucleic acid extraction cassette suitable for operation in a low resource setting. This simple design contains processing solutions preloaded within a continuous length of 1.6 mm inner diameter Tygon tubing. Processing solutions are separated by air gaps and held in place during processing by the surface tension forces at the liquid-air interface, viz. surface tension valves. Nucleic acids preferentially adsorbed to silica-coated magnetic particles are separated from sample interferents by using an external magnet to transfer the nucleic acid biomarker through successive solutions to precipitate, wash and elute in the final cassette solution. The efficiency of the extraction cassette was evaluated using quantitative reverse transcriptase PCR (qRT-PCR) following extraction of respiratory syncytial virus (RSV) RNA. RNA was recovered from TE buffer or from lysates of RSV infected HEp-2 cells with 55 and 33% efficiency, respectively, of the Qiagen RNeasy kit. Recovery of RSV RNA from RSV infected HEp-2 cells was similar at 30% of the RNeasy kit. An overall limit of detection after extraction was determined to be nearly identical (97.5%) to a laboratory-based commercially available kit. These results indicate that this extraction cassette design has the potential to be an effective sample preparation device suitable for use in a low resource setting. PMID:21604768

  14. OPTIMISATION OF TOTAL RNA EXTRACTION FROM BOVINE OOCYTES AND EMBRYOS FOR GENE EXPRESSION STUDIES AND EFFECTS OF CRYOPROTECTANTS ON TOTAL RNA EXTRACTION.

    PubMed

    Pavani, K C; Baron, E E; Faheem, M; Chaveiro, A; Da Silva, F Moreira

    2015-01-01

    Gene expression is required for understanding bovine oocytes meiotic maturation as well as the potential of embryonic development. In the present study a standardized reagent protocol for total RNA extraction was designed for bovine oocytes and embryos, which is considered specific and less expensive. For such purpose oocytes (n = 795) recovered from about 80 ovaries were divided in three groups: Group 1 modified Trizol (MTP, n = 355); Group 2 Guanidinium thiocyanate protocol (GNTC, n = 140) and Group 3 Commercial Kit protocol (CKP, n = 60). Oocytes belonging to group 1 (n = 100) and 3 (n = 20) were subjected to vitrification using two cryoprotectants 1,2 propandiol (PROH) or Dimethylsulfoxide (DMSO). The 240 remaining oocytes were divided into 3 groups in which 100 were used, in fresh, for in vitro fertilization, and 140 oocytes were vitrified using PROH (n = 70) and DMSO (n = 70) as cryoprotectants, being then fertilized in vitro after thawing. Embryos were used nine days after fertilization. Gene amplification (SDHA, (GAPDH and DNMT1) was performed in oocytes, and gene quantification (DNMT1) in in vitro produced embryos at the stage of blastocyst (n = 10). Efficiency of the extraction was further compared. The purity of all samples to different protocols ranged from 1.10 to 1.25 for GNTC protocol; from 2.05 to 2.63 for the CKP and from 1.50 to 2.11 for the developed MTP, being the last one nearest to the expected purity levels for RNA samples (1.7 to 2.0). On average, for 30 fresh oocytes, from spectrophotometer readings, total RNA concentration was 127.8 ± 9.3 ng μl(-1) for MTP, against 46.4 ± 9.5 ng μl(-1) from CKP and 476 ± 12.9 ng μl(-1) for GNTC protocol. Using the MTP to evaluate RNA in 30 vitrified/thawed oocytes, resulted in a total RNA concentration of 61.3 ± 3.3 ng μl(-1) and 40.0 μ 12.4 ng μ(-1), respectively for DMSO and PROH. Regarding total RNA concentration and purity, in blastocyst stage, more purity was observed in DMSO as compared to

  15. Using a commercial DNA extraction kit to obtain RNA for RT-PCR from starchy rice endosperm.

    PubMed

    Belefant-Miller, Helen; Ledbetter, Cindy; Bennett, Selester

    2008-03-01

    The extraction of RNA from a starchy plant material, such as many common food grains, is difficult, and especially so from the mature endosperm of rice. Most commercial RNA kits are not suitable for starchy materials. Traditional RNA extraction procedures, in addition to being laborious and time consuming, leave hazardous organic wastes that result in expensive disposal costs. Interestingly, the numerous commercial DNA isolation kits now available often include directions for eliminating co-isolated RNA. This indicated an approach to obtain the generally unwanted RNA by-product by treating the total extraction product to intentionally retain RNA. A method was developed by which a two-step DNase procedure was applied to the product of the Cartagen Food DNA extraction kit that eliminated the DNA but left the co-extracted RNA. This modified procedure was compared with several other commercial and standard methods that are promoted as being able to work under high polysaccharide conditions. Successful extraction was determined by the production and amplification of cDNA by RT-PCR of actin. Extraction was successful from milled rice, as well as from cornmeal and wheat flour. The modification provides an RNA extraction method that is quick, easy, and inexpensive, and also eliminates the production of hazardous wastes.

  16. A high-throughput RNA extraction for sprouted single-seed malting barley (Hordeum vulgare L.) rich in polysaccharides

    USDA-ARS?s Scientific Manuscript database

    Germinated seed from cereal crops including barley (Hordeum vulgare L.) is an important tissue to extract RNA and analyze expression levels of genes that control aspects of germination. These tissues are rich in polysaccharides and most methods for RNA extraction are not suitable to handle the exces...

  17. A novel automated device for rapid nucleic acid extraction utilizing a zigzag motion of magnetic silica beads.

    PubMed

    Yamaguchi, Akemi; Matsuda, Kazuyuki; Uehara, Masayuki; Honda, Takayuki; Saito, Yasunori

    2016-02-04

    We report a novel automated device for nucleic acid extraction, which consists of a mechanical control system and a disposable cassette. The cassette is composed of a bottle, a capillary tube, and a chamber. After sample injection in the bottle, the sample is lysed, and nucleic acids are adsorbed on the surface of magnetic silica beads. These magnetic beads are transported and are vibrated through the washing reagents in the capillary tube under the control of the mechanical control system, and thus, the nucleic acid is purified without centrifugation. The purified nucleic acid is automatically extracted in 3 min for the polymerase chain reaction (PCR). The nucleic acid extraction is dependent on the transport speed and the vibration frequency of the magnetic beads, and optimizing these two parameters provided better PCR efficiency than the conventional manual procedure. There was no difference between the detection limits of our novel device and that of the conventional manual procedure. We have already developed the droplet-PCR machine, which can amplify and detect specific nucleic acids rapidly and automatically. Connecting the droplet-PCR machine to our novel automated extraction device enables PCR analysis within 15 min, and this system can be made available as a point-of-care testing in clinics as well as general hospitals.

  18. A System for Automated Extraction of Metadata from Scanned Documents using Layout Recognition and String Pattern Search Models.

    PubMed

    Misra, Dharitri; Chen, Siyuan; Thoma, George R

    2009-01-01

    One of the most expensive aspects of archiving digital documents is the manual acquisition of context-sensitive metadata useful for the subsequent discovery of, and access to, the archived items. For certain types of textual documents, such as journal articles, pamphlets, official government records, etc., where the metadata is contained within the body of the documents, a cost effective method is to identify and extract the metadata in an automated way, applying machine learning and string pattern search techniques.At the U. S. National Library of Medicine (NLM) we have developed an automated metadata extraction (AME) system that employs layout classification and recognition models with a metadata pattern search model for a text corpus with structured or semi-structured information. A combination of Support Vector Machine and Hidden Markov Model is used to create the layout recognition models from a training set of the corpus, following which a rule-based metadata search model is used to extract the embedded metadata by analyzing the string patterns within and surrounding each field in the recognized layouts.In this paper, we describe the design of our AME system, with focus on the metadata search model. We present the extraction results for a historic collection from the Food and Drug Administration, and outline how the system may be adapted for similar collections. Finally, we discuss some ongoing enhancements to our AME system.

  19. A System for Automated Extraction of Metadata from Scanned Documents using Layout Recognition and String Pattern Search Models

    PubMed Central

    Misra, Dharitri; Chen, Siyuan; Thoma, George R.

    2010-01-01

    One of the most expensive aspects of archiving digital documents is the manual acquisition of context-sensitive metadata useful for the subsequent discovery of, and access to, the archived items. For certain types of textual documents, such as journal articles, pamphlets, official government records, etc., where the metadata is contained within the body of the documents, a cost effective method is to identify and extract the metadata in an automated way, applying machine learning and string pattern search techniques. At the U. S. National Library of Medicine (NLM) we have developed an automated metadata extraction (AME) system that employs layout classification and recognition models with a metadata pattern search model for a text corpus with structured or semi-structured information. A combination of Support Vector Machine and Hidden Markov Model is used to create the layout recognition models from a training set of the corpus, following which a rule-based metadata search model is used to extract the embedded metadata by analyzing the string patterns within and surrounding each field in the recognized layouts. In this paper, we describe the design of our AME system, with focus on the metadata search model. We present the extraction results for a historic collection from the Food and Drug Administration, and outline how the system may be adapted for similar collections. Finally, we discuss some ongoing enhancements to our AME system. PMID:21179386

  20. RNA Chromogenic in situ Hybridization Assay with Clinical Automated Platform is a Sensitive Method in Detecting High-risk Human Papillomavirus in Squamous Cell Carcinoma.

    PubMed

    Mendez-Pena, Javier E; Sadow, Peter M; Nose, Vania; Hoang, Mai P

    2017-03-13

    Detection of active human papillomavirus (HPV) is clinically important, as its presence has been shown to correlate with favorable clinical outcomes and better response to treatment in oropharyngeal squamous cell carcinomas (SCC). Using a clinical automated platform, we compared the performance of commercially available HPV DNA and RNA in situ hybridization (ISH) probes in archival tissues of 57 SCC. Importantly, a clinical automated platform gives 1) consistent and reproducible results for HPV ISH and 2) better standardization across clinical laboratories. Compared to polymerase chain reaction (PCR) results, RNA ISH exhibited 93% concordance versus 81% of DNA ISH. RNA ISH was more sensitive than DNA ISH (100% versus 88%), and more specific (87% versus 74%). When only accounting for 2-3+ positivity, sensitivity was 92% for RNA ISH versus 73% for DNA ISH, highlighting the ease of interpretation. p16 exhibited 96% sensitivity while specificity was only 55%. In 3 cases both RNA and DNA ISH were positive while PCR results were negative, suggesting that ISH methods might be a more sensitive method. Performing on a clinical automated platform, RNA ISH is sensitive in determining high-risk HPV status in formalin-fixed paraffin-embedded tissues and has the potential of being a standalone clinical test.

  1. RNA sequencing using fluorescent-labeled dideoxynucleotides and automated fluorescence detection.

    PubMed Central

    Bauer, G J

    1990-01-01

    Although dideoxy terminated sequencing of RNA, using reverse transcriptase and oligodeoxynucleotide primers, is now a well established method, the accuracy is limited by sequence ambiguities due to unspecific chain termination events. A protocol is described which circumvents these ambiguities by using fluorescence labels tagged to dideoxynucleotides. Only chain terminations caused by dideoxynucleotides were detected while premature terminated cDNA's remain undetectable. In addition, the remaining multiple signals at nucleotide positions can be assigned to sequence heterogeneities within the RNA sequence to be determined. Images PMID:1690393

  2. miRTex: A Text Mining System for miRNA-Gene Relation Extraction.

    PubMed

    Li, Gang; Ross, Karen E; Arighi, Cecilia N; Peng, Yifan; Wu, Cathy H; Vijay-Shanker, K

    2015-01-01

    MicroRNAs (miRNAs) regulate a wide range of cellular and developmental processes through gene expression suppression or mRNA degradation. Experimentally validated miRNA gene targets are often reported in the literature. In this paper, we describe miRTex, a text mining system that extracts miRNA-target relations, as well as miRNA-gene and gene-miRNA regulation relations. The system achieves good precision and recall when evaluated on a literature corpus of 150 abstracts with F-scores close to 0.90 on the three different types of relations. We conducted full-scale text mining using miRTex to process all the Medline abstracts and all the full-length articles in the PubMed Central Open Access Subset. The results for all the Medline abstracts are stored in a database for interactive query and file download via the website at http://proteininformationresource.org/mirtex. Using miRTex, we identified genes potentially regulated by miRNAs in Triple Negative Breast Cancer, as well as miRNA-gene relations that, in conjunction with kinase-substrate relations, regulate the response to abiotic stress in Arabidopsis thaliana. These two use cases demonstrate the usefulness of miRTex text mining in the analysis of miRNA-regulated biological processes.

  3. miRTex: A Text Mining System for miRNA-Gene Relation Extraction

    PubMed Central

    Li, Gang; Ross, Karen E.; Arighi, Cecilia N.; Peng, Yifan; Wu, Cathy H.; Vijay-Shanker, K.

    2015-01-01

    MicroRNAs (miRNAs) regulate a wide range of cellular and developmental processes through gene expression suppression or mRNA degradation. Experimentally validated miRNA gene targets are often reported in the literature. In this paper, we describe miRTex, a text mining system that extracts miRNA-target relations, as well as miRNA-gene and gene-miRNA regulation relations. The system achieves good precision and recall when evaluated on a literature corpus of 150 abstracts with F-scores close to 0.90 on the three different types of relations. We conducted full-scale text mining using miRTex to process all the Medline abstracts and all the full-length articles in the PubMed Central Open Access Subset. The results for all the Medline abstracts are stored in a database for interactive query and file download via the website at http://proteininformationresource.org/mirtex. Using miRTex, we identified genes potentially regulated by miRNAs in Triple Negative Breast Cancer, as well as miRNA-gene relations that, in conjunction with kinase-substrate relations, regulate the response to abiotic stress in Arabidopsis thaliana. These two use cases demonstrate the usefulness of miRTex text mining in the analysis of miRNA-regulated biological processes. PMID:26407127

  4. Inhibition of RNA polymerase II transcription in human cell extracts by cisplatin DNA damage.

    PubMed

    Cullinane, C; Mazur, S J; Essigmann, J M; Phillips, D R; Bohr, V A

    1999-05-11

    The anticancer drug cisplatin induces a spectrum of lesions in DNA. The effect of such DNA damage on transcription by RNA polymerase II (RNA pol II) in human cell extracts was investigated at the level of initiation and elongation. RNA pol II transcription directed from the adenovirus major late promoter was inhibited following treatment of the promoter-containing template with increasing concentrations of cisplatin. Furthermore, transcription from an undamaged promoter fragment was depleted in the presence of increasing amounts of cisplatin DNA damage on an exogenous plasmid, suggesting such damage may hijack an essential factor for transcription initiation. The effect of cisplatin damage on RNA pol II elongation was investigated using site-specifically-placed cisplatin adducts. The GTG adduct was an effective block to RNA pol II elongation, inhibiting the polymerase by 80%. In contrast, RNA pol II completely bypassed the cisplatin GG intrastrand adduct. These studies suggest that the inhibition of RNA pol II transcription observed following the treatment of cells with cisplatin is likely to reflect the combined effects of DNA damage at the level of both transcription initiation and elongation.

  5. A High-Throughput RNA Extraction for Sprouted Single-Seed Barley (Hordeum vulgare L.) Rich in Polysaccharides

    PubMed Central

    Rashid, Abdur; Baldwin, Thomas; Gines, Michael; Bregitzer, Phil; Esvelt Klos, Kathy

    2016-01-01

    Germinated seed from cereal crops including barley (Hordeum vulgare L.) is an important tissue to extract RNA and analyze expression levels of genes that control aspects of germination. These tissues are rich in polysaccharides and most methods for RNA extraction are not suitable to handle the excess polysaccharides. Here, we compare the current methods for RNA extraction applicable to germinated barley tissue. We found that although some of these standard methods produced high-quality RNA, the process of extraction was drastically slow, mostly because the frozen seed tissue powder from liquid N2 grinding became recalcitrant to buffer mixing. Our suggested modifications to the protocols removed the need for liquid N2 grinding and significantly increased the output efficiency of RNA extraction. Our modified protocol has applications in other cereal tissues rich in polysaccharides, including oat. PMID:28025509

  6. A High-Throughput RNA Extraction for Sprouted Single-Seed Barley (Hordeum vulgare L.) Rich in Polysaccharides.

    PubMed

    Rashid, Abdur; Baldwin, Thomas; Gines, Michael; Bregitzer, Phil; Esvelt Klos, Kathy

    2016-12-22

    Germinated seed from cereal crops including barley (Hordeum vulgare L.) is an important tissue to extract RNA and analyze expression levels of genes that control aspects of germination. These tissues are rich in polysaccharides and most methods for RNA extraction are not suitable to handle the excess polysaccharides. Here, we compare the current methods for RNA extraction applicable to germinated barley tissue. We found that although some of these standard methods produced high-quality RNA, the process of extraction was drastically slow, mostly because the frozen seed tissue powder from liquid N₂ grinding became recalcitrant to buffer mixing. Our suggested modifications to the protocols removed the need for liquid N₂ grinding and significantly increased the output efficiency of RNA extraction. Our modified protocol has applications in other cereal tissues rich in polysaccharides, including oat.

  7. Mixed-mode isolation of triazine metabolites from soil and aquifer sediments using automated solid-phase extraction

    USGS Publications Warehouse

    Mills, M.S.; Thurman, E.M.

    1992-01-01

    Reversed-phase isolation and ion-exchange purification were combined in the automated solid-phase extraction of two polar s-triazine metabolites, 2-amino-4-chloro-6-(isopropylamino)-s-triazine (deethylatrazine) and 2-amino-4-chloro-6-(ethylamino)-s-triazine (deisopropylatrazine) from clay-loam and slit-loam soils and sandy aquifer sediments. First, methanol/ water (4/1, v/v) soil extracts were transferred to an automated workstation following evaporation of the methanol phase for the rapid reversed-phase isolation of the metabolites on an octadecylresin (C18). The retention of the triazine metabolites on C18 decreased substantially when trace methanol concentrations (1%) remained. Furthermore, the retention on C18 increased with decreasing aqueous solubility and increasing alkyl-chain length of the metabolites and parent herbicides, indicating a reversed-phase interaction. The analytes were eluted with ethyl acetate, which left much of the soil organic-matter impurities on the resin. Second, the small-volume organic eluate was purified on an anion-exchange resin (0.5 mL/min) to extract the remaining soil pigments that could foul the ion source of the GC/MS system. Recoveries of the analytes were 75%, using deuterated atrazine as a surrogate, and were comparable to recoveries by soxhlet extraction. The detection limit was 0.1 ??g/kg with a coefficient of variation of 15%. The ease and efficiency of this automated method makes it viable, practical technique for studying triazine metabolites in the environment.

  8. Method for RNA extraction and cDNA library construction from microbes in crop rhizosphere soil.

    PubMed

    Fang, Changxun; Xu, Tiecheng; Ye, Changliang; Huang, Likun; Wang, Qingshui; Lin, Wenxiong

    2014-02-01

    Techniques to analyze the transcriptome of the soil rhizosphere are essential to reveal the interactions and communications between plants and microorganisms in the soil ecosystem. In this study, different volumes of Al₂(SO₄)₃ were added to rhizosphere soil samples to precipitate humic substances, which interfere with most procedures of RNA and DNA analyses. After humic substances were precipitated, cells of soil microorganisms were broken by vortexing with glass beads, and then DNA and RNA were recovered using Tris-HCl buffer with LiCl, SDS, and EDTA. The crude extract was precipitated and dissolved in RNAse-free water, and then separated by agarose gel electrophoresis. We determined the optimum volume of Al₂(SO₄)₃ for treating rhizosphere soil of rice, tobacco, sugarcane, Rehmannia glutinosa, and Pseudostellaria heterophylla. The crude nucleic acids extract from rice soil was treated with DNase I and then RNA was purified using a gel filtration column. The purified RNA was reverse-transcribed into single-strand cDNA and then ligated with an adaptor at each end before amplifying ds cDNA. The ds cDNA was sub-cloned for subsequent gene sequence analysis. We conducted qPCR to amplify 16S ribosomal DNA and observed highly efficient amplification. These results show that the extraction method can be optimized to isolate and obtain high-quality nucleic acids from microbes in different rhizosphere soils, suitable for genomic and post-genomic analyses.

  9. High quality RNA extraction of the mammalian cochlea for qRT-PCR and transcriptome analyses.

    PubMed

    Vikhe Patil, Kim; Canlon, Barbara; Cederroth, Christopher R

    2015-07-01

    Molecular investigations of the hearing organ, the cochlea, have been hampered due to the difficulty of isolating pure RNA and in quantities sufficient enough for quantitative real-time RT-PCR or microarray analysis. The complex architecture of the cochlea, the presence of liquids, bone and cartilage tissue, are a major hurdle in obtaining contamination-free RNA to a level that does not affect downstream applications. Here, we present a protocol to extract RNA from the mouse cochlea, with yields and quality suitable for real-time RT-PCR or Affymetrix labeling. In contrast to current methods, such as TRIZOL or column-based extraction, this protocol combines the two and, within 4 h, yields a 2 μg of total RNA from a single pair of adult mouse cochleae. This protocol allows the isolation of RNA molecules from the mammalian cochlea providing access to whole-transcript expression analyses. Copyright © 2015 Elsevier B.V. All rights reserved.

  10. Extracting microRNA-gene relations from biomedical literature using distant supervision

    PubMed Central

    Clarke, Luka A.; Couto, Francisco M.

    2017-01-01

    Many biomedical relation extraction approaches are based on supervised machine learning, requiring an annotated corpus. Distant supervision aims at training a classifier by combining a knowledge base with a corpus, reducing the amount of manual effort necessary. This is particularly useful for biomedicine because many databases and ontologies have been made available for many biological processes, while the availability of annotated corpora is still limited. We studied the extraction of microRNA-gene relations from text. MicroRNA regulation is an important biological process due to its close association with human diseases. The proposed method, IBRel, is based on distantly supervised multi-instance learning. We evaluated IBRel on three datasets, and the results were compared with a co-occurrence approach as well as a supervised machine learning algorithm. While supervised learning outperformed on two of those datasets, IBRel obtained an F-score 28.3 percentage points higher on the dataset for which there was no training set developed specifically. To demonstrate the applicability of IBRel, we used it to extract 27 miRNA-gene relations from recently published papers about cystic fibrosis. Our results demonstrate that our method can be successfully used to extract relations from literature about a biological process without an annotated corpus. The source code and data used in this study are available at https://github.com/AndreLamurias/IBRel. PMID:28263989

  11. Extracting microRNA-gene relations from biomedical literature using distant supervision.

    PubMed

    Lamurias, Andre; Clarke, Luka A; Couto, Francisco M

    2017-01-01

    Many biomedical relation extraction approaches are based on supervised machine learning, requiring an annotated corpus. Distant supervision aims at training a classifier by combining a knowledge base with a corpus, reducing the amount of manual effort necessary. This is particularly useful for biomedicine because many databases and ontologies have been made available for many biological processes, while the availability of annotated corpora is still limited. We studied the extraction of microRNA-gene relations from text. MicroRNA regulation is an important biological process due to its close association with human diseases. The proposed method, IBRel, is based on distantly supervised multi-instance learning. We evaluated IBRel on three datasets, and the results were compared with a co-occurrence approach as well as a supervised machine learning algorithm. While supervised learning outperformed on two of those datasets, IBRel obtained an F-score 28.3 percentage points higher on the dataset for which there was no training set developed specifically. To demonstrate the applicability of IBRel, we used it to extract 27 miRNA-gene relations from recently published papers about cystic fibrosis. Our results demonstrate that our method can be successfully used to extract relations from literature about a biological process without an annotated corpus. The source code and data used in this study are available at https://github.com/AndreLamurias/IBRel.

  12. Transcriptator: An Automated Computational Pipeline to Annotate Assembled Reads and Identify Non Coding RNA.

    PubMed

    Tripathi, Kumar Parijat; Evangelista, Daniela; Zuccaro, Antonio; Guarracino, Mario Rosario

    2015-01-01

    RNA-seq is a new tool to measure RNA transcript counts, using high-throughput sequencing at an extraordinary accuracy. It provides quantitative means to explore the transcriptome of an organism of interest. However, interpreting this extremely large data into biological knowledge is a problem, and biologist-friendly tools are lacking. In our lab, we developed Transcriptator, a web application based on a computational Python pipeline with a user-friendly Java interface. This pipeline uses the web services available for BLAST (Basis Local Search Alignment Tool), QuickGO and DAVID (Database for Annotation, Visualization and Integrated Discovery) tools. It offers a report on statistical analysis of functional and Gene Ontology (GO) annotation's enrichment. It helps users to identify enriched biological themes, particularly GO terms, pathways, domains, gene/proteins features and protein-protein interactions related informations. It clusters the transcripts based on functional annotations and generates a tabular report for functional and gene ontology annotations for each submitted transcript to the web server. The implementation of QuickGo web-services in our pipeline enable the users to carry out GO-Slim analysis, whereas the integration of PORTRAIT (Prediction of transcriptomic non coding RNA (ncRNA) by ab initio methods) helps to identify the non coding RNAs and their regulatory role in transcriptome. In summary, Transcriptator is a useful software for both NGS and array data. It helps the users to characterize the de-novo assembled reads, obtained from NGS experiments for non-referenced organisms, while it also performs the functional enrichment analysis of differentially expressed transcripts/genes for both RNA-seq and micro-array experiments. It generates easy to read tables and interactive charts for better understanding of the data. The pipeline is modular in nature, and provides an opportunity to add new plugins in the future. Web application is freely

  13. A streamlined protocol for extracting RNA and genomic DNA from archived human blood and muscle.

    PubMed

    Majumdar, Gipsy; Vera, Santiago; Elam, Marshall B; Raghow, Rajendra

    2015-04-01

    We combined the TRIzol method of nucleic acid extraction with QIAamp columns to achieve coextraction of RNA and genomic DNA from peripheral blood mononuclear cells (PBMCs) and biopsied skeletal muscle, both stored at -80 °C for many months. Total RNA was recovered from the upper aqueous phase of TRIzol. The interphase and organic phases were precipitated with ethanol, digested with proteinase K, and filtered through QIAamp MinElute columns to recover DNA. The combined protocol yielded excellent quality and quantity of nucleic acids from archived human PBMCs and muscle and may be easily adapted for other tissues. Published by Elsevier Inc.

  14. A Modified Protocol for High-Quality RNA Extraction from Oleoresin-Producing Adult Pines.

    PubMed

    de Lima, Júlio César; Füller, Thanise Nogueira; de Costa, Fernanda; Rodrigues-Corrêa, Kelly C S; Fett-Neto, Arthur G

    2016-01-01

    RNA extraction resulting in good yields and quality is a fundamental step for the analyses of transcriptomes through high-throughput sequencing technologies, microarray, and also northern blots, RT-PCR, and RTqPCR. Even though many specific protocols designed for plants with high content of secondary metabolites have been developed, these are often expensive, time consuming, and not suitable for a wide range of tissues. Here we present a modification of the method previously described using the commercially available Concert™ Plant RNA Reagent (Invitrogen) buffer for field-grown adult pine trees with high oleoresin content.

  15. 16S rRNA Gene Sequence Analysis of Drinking Water Using RNA and DNA Extracts as Targets for Clone Library Development - Poster

    EPA Science Inventory

    We examined the bacterial composition of chlorinated drinking water using 16S rRNA gene clone libraries derived from RNA and DNA extracted from twelve water samples collected in three different months (June, August, and September of 2007). Phylogenetic analysis of 1234 and 1117 ...

  16. 16S rRNA Gene Sequence Analysis of Drinking Water Using RNA and DNA Extracts as Targets for Clone Library Development

    EPA Science Inventory

    We examined the bacterial composition of chlorinated drinking water using 16S rRNA gene clone libraries derived from RNA and DNA extracted from twelve water samples collected in three different months (June, August, and September of 2007). Phylogenetic analysis of 1234 and 1117 ...

  17. 16S rRNA Gene Sequence Analysis of Drinking Water Using RNA and DNA Extracts as Targets for Clone Library Development

    EPA Science Inventory

    We examined the bacterial composition of chlorinated drinking water using 16S rRNA gene clone libraries derived from RNA and DNA extracted from twelve water samples collected in three different months (June, August, and September of 2007). Phylogenetic analysis of 1234 and 1117 ...

  18. 16S rRNA Gene Sequence Analysis of Drinking Water Using RNA and DNA Extracts as Targets for Clone Library Development - Poster

    EPA Science Inventory

    We examined the bacterial composition of chlorinated drinking water using 16S rRNA gene clone libraries derived from RNA and DNA extracted from twelve water samples collected in three different months (June, August, and September of 2007). Phylogenetic analysis of 1234 and 1117 ...

  19. Highly integrated flow assembly for automated dynamic extraction and determination of readily bioaccessible chromium(VI) in soils exploiting carbon nanoparticle-based solid-phase extraction.

    PubMed

    Rosende, María; Miró, Manuel; Segundo, Marcela A; Lima, José L F C; Cerdà, Víctor

    2011-06-01

    An automated dynamic leaching test integrated in a portable flow-based setup is herein proposed for reliable determination of readily bioaccessible Cr(VI) under worst-case scenarios in soils containing varying levels of contamination. The manifold is devised to accommodate bi-directional flow extraction followed by processing of extracts via either in-line clean-up/preconcentration using multi-walled carbon nanotubes or automatic dilution at will, along with Cr(VI) derivatization and flow-through spectrophotometric detection. The magnitude of readily mobilizable Cr(VI) pools was ascertained by resorting to water extraction as promulgated by current standard leaching tests. The role of carbon nanomaterials for the uptake of Cr(VI) in soil leachates and the configuration of the packed column integrated in the flow manifold were investigated in detail. The analytical performance of the proposed system for in vitro bioaccessibility tests was evaluated in chromium-enriched soils at environmentally relevant levels and in a standard reference soil material (SRM 2701) with a certified value of total hexavalent chromium. The automated method was proven to afford unbiased assessment of water-soluble Cr(VI) in soils as a result of the minimization of the chromium species transformation. By combination of the kinetic leaching profile and a first-order leaching model, the water-soluble Cr(VI) fraction in soils was determined in merely 6 h against >24 h taken in batchwise steady-state standard methods.

  20. Optimizing preservation protocols to extract high-quality RNA from different tissues of echinoderms for next-generation sequencing.

    PubMed

    Pérez-Portela, Rocío; Riesgo, Ana

    2013-09-01

    Transcriptomic information provides fundamental insights into biological processes. Extraction of quality RNA is a challenging step, and preservation and extraction protocols need to be adjusted in many cases. Our objectives were to optimize preservation protocols for isolation of high-quality RNA from diverse echinoderm tissues and to compare the utility of parameters as absorbance ratios and RIN values to assess RNA quality. Three different tissues (gonad, oesophagus and coelomocytes) were selected from the sea urchin Arbacia lixula. Solid tissues were flash-frozen and stored at -80 °C until processed. Four preservation treatments were applied to coelomocytes: flash freezing and storage at -80 °C, RNAlater and storage at -20 °C, preservation in TRIzol reagent and storage at -80 °C and direct extraction with TRIzol from fresh cells. Extractions of total RNA were performed with a modified TRIzol protocol for all tissues. Our results showed high values of RNA quantity and quality for all tissues, showing nonsignificant differences among them. However, while flash freezing was effective for solid tissues, it was inadequate for coelomocytes because of the low quality of the RNA extractions. Coelomocytes preserved in RNAlater displayed large variability in RNA integrity and insufficient RNA amount for further isolation of mRNA. TRIzol was the most efficient system for stabilizing RNA which resulted on high RNA quality and quantity. We did not detect correlation between absorbance ratios and RNA integrity. The best strategies for assessing RNA integrity was the visualization of 18S rRNA and 28S rRNA bands in agarose gels and estimation of RIN values with Agilent Bioanalyzer chips. © 2013 John Wiley & Sons Ltd.

  1. High-yield extraction of Escherichia coli RNA from human whole blood.

    PubMed

    Brennecke, Johannes; Kraut, Simone; Zwadlo, Klara; Gandi, Senthil Kumar; Pritchard, David; Templeton, Kate; Bachmann, Till

    2017-03-01

    Studies of bacterial transcriptomics during bloodstream infections are limited to-date because unbiased extraction of bacterial mRNA from whole blood in sufficient quantity and quality has proved challenging. Problems include the high excess of human cells, the presence of PCR inhibitors and the short intrinsic half-life of bacterial mRNA. This study aims to provide a framework for the choice of the most suitable sample preparation method. Escherichia coli cells were spiked into human whole blood and the bacterial gene expression was stabilized with RNAprotect either immediately or after lysis of the red blood cells with Triton X-100, saponin, ammonium chloride or the commercial MolYsis buffer CM. RNA yield, purity and integrity were assessed by absorbance measurements at 260 and 280 nm, real-time PCR and capillary electrophoresis. For low cell numbers, the best mRNA yields were obtained by adding the commercial RNAprotect reagent directly to the sample without prior lyses of the human blood cells. Using this protocol, significant amounts of human RNA were co-purified, however, this had a beneficial impact on the yields of bacterial mRNA. Among the tested lysis agents, Triton X-100 was the most effective and reduced the human RNA background by three to four orders of magnitude. For most applications, lysis of the human blood cells is not required. However, co-purified human RNA may interfere with some downstream processes such as RNA sequencing. In this case, blood cell lysis with Triton X-100 is desirable.

  2. Semi-automated extraction of landslides in Taiwan based on SPOT imagery and DEMs

    NASA Astrophysics Data System (ADS)

    Eisank, Clemens; Hölbling, Daniel; Friedl, Barbara; Chen, Yi-Chin; Chang, Kang-Tsung

    2014-05-01

    The vast availability and improved quality of optical satellite data and digital elevation models (DEMs), as well as the need for complete and up-to-date landslide inventories at various spatial scales have fostered the development of semi-automated landslide recognition systems. Among the tested approaches for designing such systems, object-based image analysis (OBIA) stepped out to be a highly promising methodology. OBIA offers a flexible, spatially enabled framework for effective landslide mapping. Most object-based landslide mapping systems, however, have been tailored to specific, mainly small-scale study areas or even to single landslides only. Even though reported mapping accuracies tend to be higher than for pixel-based approaches, accuracy values are still relatively low and depend on the particular study. There is still room to improve the applicability and objectivity of object-based landslide mapping systems. The presented study aims at developing a knowledge-based landslide mapping system implemented in an OBIA environment, i.e. Trimble eCognition. In comparison to previous knowledge-based approaches, the classification of segmentation-derived multi-scale image objects relies on digital landslide signatures. These signatures hold the common operational knowledge on digital landslide mapping, as reported by 25 Taiwanese landslide experts during personal semi-structured interviews. Specifically, the signatures include information on commonly used data layers, spectral and spatial features, and feature thresholds. The signatures guide the selection and implementation of mapping rules that were finally encoded in Cognition Network Language (CNL). Multi-scale image segmentation is optimized by using the improved Estimation of Scale Parameter (ESP) tool. The approach described above is developed and tested for mapping landslides in a sub-region of the Baichi catchment in Northern Taiwan based on SPOT imagery and a high-resolution DEM. An object

  3. mRNA-specific reverse transcription-polymerase chain reaction from human tissue extracts.

    PubMed

    Hurteau, Gregory J; Spivack, Simon D

    2002-08-15

    Reverse transcription-polymerase chain reaction (RT-PCR) has become the method of choice for detection of mRNA transcripts, including those of low abundance obtained from small precious samples of human tissue. A major confounding problem for standard reverse-transcription-priming strategies is the presence of contaminating genomic DNA (gDNA) carried over from the original "RNA" extract into the RT and PCR steps. The contaminating gDNA contains a processed pseudogene sequence-which lacks introns but contains a poly(A) tail-for commonly studied internal reference genes beta-actin and GAPDH, and target genes GSTM1, GSTP1, and others. These pseudogene sequences therefore confound standard-design "RNA-specific" PCR primer pairs which rely, for cDNA versus gDNA specificity, on the pair-spanning introns, or one of the individual primer oligos spanning an exon/exon splice site, because these features are lacking in processed pseudogene sequences. The result is false RT-PCR positives for these "housekeeper" genes in total RNA extracts; the gDNA processed pseudogene is mistaken for mRNA gene transcript. A universal RT primer has been designed that targets the poly(A) tail of mRNA and adds a unique tag sequence not otherwise existing in the human genome. Genomic DNA does not incorporate this RT-inserted unique tag. PCR is then performed using a transcript-specific forward primer and a reverse primer that is identical to the unique tag incorporated at RT. Only cDNA made with this RT primer is compatible with this reverse PCR primer, thus eliminating confounding signal from contaminating gDNA. This method performs RNA-specific qualitative and quantitative evaluation of gene expression, while preserving the sensitivity of standard RT-PCR techniques. Applications to low-copy transcripts in human samples are demonstrated.

  4. EPIG-Seq: extracting patterns and identifying co-expressed genes from RNA-Seq data.

    PubMed

    Li, Jianying; Bushel, Pierre R

    2016-03-22

    RNA sequencing (RNA-Seq) measures genome-wide gene expression. RNA-Seq data is count-based rendering normal distribution models for analysis inappropriate. Normalization of RNA-Seq data to transform the data has limitations which can adversely impact the analysis. Furthermore, there are a few count-based methods for analysis of RNA-Seq data but they are essentially for pairwise analysis of treatment groups or multiclasses but not pattern-based to identify co-expressed genes. We adapted our extracting patterns and identifying genes methodology for RNA-Seq (EPIG-Seq) count data. The software uses count-based correlation to measure similarity between genes, quasi-Poisson modelling to estimate dispersion in the data and a location parameter to indicate magnitude of differential expression. EPIG-Seq is different than any other software currently available for pattern analysis of RNA-Seq data in that EPIG-Seq 1) uses count level data and supports cases of inflated zeros, 2) identifies statistically significant clusters of genes that are co-expressed across experimental conditions, 3) takes into account dispersion in the replicate data and 4) provides reliable results even with small sample sizes. EPIG-Seq operates in two steps: 1) extract the pattern profiles from data as seeds for clustering co-expressed genes and 2) cluster the genes to the pattern seeds and compute statistical significance of the pattern of co-expressed genes. EPIG-Seq provides a table of the genes with bootstrapped p-values and profile plots of the patterns of co-expressed genes. In addition, EPIG-Seq provides a heat map and principal component dimension reduction plot of the clustered genes as visual aids. We demonstrate the utility of EPIG-Seq through the analysis of toxicogenomics and cancer data sets to identify biologically relevant co-expressed genes. EPIG-Seq is available at: sourceforge.net/projects/epig-seq. EPIG-Seq is unlike any other software currently available for pattern analysis of

  5. Metal-organic framework mixed-matrix disks: Versatile supports for automated solid-phase extraction prior to chromatographic separation.

    PubMed

    Ghani, Milad; Font Picó, Maria Francesca; Salehinia, Shima; Palomino Cabello, Carlos; Maya, Fernando; Berlier, Gloria; Saraji, Mohammad; Cerdà, Víctor; Turnes Palomino, Gemma

    2017-03-10

    We present for the first time the application of metal-organic framework (MOF) mixed-matrix disks (MMD) for the automated flow-through solid-phase extraction (SPE) of environmental pollutants. Zirconium terephthalate UiO-66 and UiO-66-NH2 MOFs with different size (90, 200 and 300nm) have been incorporated into mechanically stable polyvinylidene difluoride (PVDF) disks. The performance of the MOF-MMDs for automated SPE of seven substituted phenols prior to HPLC analysis has been evaluated using the sequential injection analysis technique. MOF-MMDs enabled the simultaneous extraction of phenols with the concomitant size exclusion of molecules of larger size. The best extraction performance was obtained using a MOF-MMD containing 90nm UiO-66-NH2 crystals. Using the selected MOF-MMD, detection limits ranging from 0.1 to 0.2μgL(-1) were obtained. Relative standard deviations ranged from 3.9 to 5.3% intra-day, and 4.7-5.7% inter-day. Membrane batch-to-batch reproducibility was from 5.2 to 6.4%. Three different groundwater samples were analyzed with the proposed method using MOF-MMDs, obtaining recoveries ranging from 90 to 98% for all tested analytes.

  6. Performance evaluation of the Maxwell 16 System for extraction of influenza virus RNA from diverse samples.

    PubMed

    Liu, Hongbo; Gan, Yan; Yang, Bo; Weng, Hui; Huang, Chunmei; Yang, Daofeng; Lei, Ping; Shen, Guanxin

    2012-01-01

    This study evaluated the performance of the Maxwell 16 System (Promega) for extraction of influenza virus (flu-v) RNA from diverse samples compared to a classical manual method (QIAamp Kit, QIAGEN). Following extraction by the two methods, all samples were analyzed by Real-time RT-PCR. Results revealed that the use of the standard Maxwell 16 protocol (Maxwell 16-S) resulted in good linearity and precision across a wide concentration range and higher sensitivity of detection from flu-v stock suspensions than the manual method. Compared with the latter method, Maxwell 16-S extracted RNA more efficiently (higher RNA yield and/or fewer PCR inhibitors) from throat swabs and bronchoalveolar lavage fluids, while both methods performed comparably on fecal samples from human and poultry in terms of overall threshold cycle values and detection rates although the Maxwell 16-S co-purified more inhibitors from fecal samples. The capacity of this system to remove inhibitors from fecal matrix was improved by using a modified Maxwell 16 protocol with a reduced sample input, which eliminated all false-negatives produced by the Maxwell 16-S. These findings suggest that the Maxwell 16 System is suitable for RNA extraction from multiple-source samples for diagnosis of influenza and viral load determination and that a proper reduction in starting sample volume may improve the detection of flu-v from complex matrices such as feces. Additionally, this system allows flexible sample throughput and labor-saving sample processing with little or no risk of cross-contamination.

  7. Performance Evaluation of the Maxwell 16 System for Extraction of Influenza Virus RNA from Diverse Samples

    PubMed Central

    Liu, Hongbo; Gan, Yan; Yang, Bo; Weng, Hui; Huang, Chunmei; Yang, Daofeng; Lei, Ping; Shen, Guanxin

    2012-01-01

    This study evaluated the performance of the Maxwell 16 System (Promega) for extraction of influenza virus (flu-v) RNA from diverse samples compared to a classical manual method (QIAamp Kit, QIAGEN). Following extraction by the two methods, all samples were analyzed by Real-time RT-PCR. Results revealed that the use of the standard Maxwell 16 protocol (Maxwell 16-S) resulted in good linearity and precision across a wide concentration range and higher sensitivity of detection from flu-v stock suspensions than the manual method. Compared with the latter method, Maxwell 16-S extracted RNA more efficiently (higher RNA yield and/or fewer PCR inhibitors) from throat swabs and bronchoalveolar lavage fluids, while both methods performed comparably on fecal samples from human and poultry in terms of overall threshold cycle values and detection rates although the Maxwell 16-S co-purified more inhibitors from fecal samples. The capacity of this system to remove inhibitors from fecal matrix was improved by using a modified Maxwell 16 protocol with a reduced sample input, which eliminated all false-negatives produced by the Maxwell 16-S. These findings suggest that the Maxwell 16 System is suitable for RNA extraction from multiple-source samples for diagnosis of influenza and viral load determination and that a proper reduction in starting sample volume may improve the detection of flu-v from complex matrices such as feces. Additionally, this system allows flexible sample throughput and labor-saving sample processing with little or no risk of cross-contamination. PMID:23144730

  8. Extraction of total RNA from a high pigment content plant. Marigold (Tagetes erecta).

    PubMed

    Chi-Manzanero, B; Robert, M L; Rivera-Madrid, R

    2000-09-01

    The Mexican marigold (Tagetes erecta) produces inflorescences of intense yellow color that contain high levels of xanthophylls, particularly lutein, which makes it a suitable model for the study of carotenoid biosynthesis and regulation throughout the development of the inflorescences. However, these studies require the recovery of total RNA from floral buds and inflorescences at different developmental stages, each of which presents specific extraction problems. Four protocols were tested, but only through the modification of one of them was it possible to obtain total RNA of sufficient quality and quantity to perform RT-PCR and Northern blots and to construct a cDNA library. This article presents the modified protocol for the recovery of total RNA from carotenoid-rich plant tissues.

  9. 16S rRNA gene sequence analysis of drinking water using RNA and DNA extracts as targets for clone library development.

    PubMed

    Revetta, Randy P; Matlib, Robin S; Santo Domingo, Jorge W

    2011-07-01

    The bacterial composition of chlorinated drinking water was analyzed using 16S rRNA gene clone libraries derived from DNA extracts of 12 samples and compared to clone libraries previously generated using RNA extracts from the same samples. Phylogenetic analysis of 761 DNA-based clone sequences showed that unclassified bacteria were the most abundant group, representing nearly 62% of all DNA sequences analyzed. Other phylogenetic groups identified included Proteobacteria (20%), Actinobacteria (9%), Cyanobacteria (4%), and Bacteroidetes (2%). The composition of RNA-based libraries (1122 sequences) was similar to the DNA-based libraries with a few notable exceptions: Proteobacteria were more dominant in the RNA clone libraries (i.e., 35% RNA; 20% DNA). Differences in the Proteobacteria composition were also observed; alpha-Proteobacteria was 22 times more abundant in the RNA-based clones while beta-Proteobacteria was eight times more abundant in the DNA libraries. Nearly twice as many DNA operational taxonomic units (OTUs) than RNA OTUs were observed at distance 0.03 (101 DNA; 53 RNA). Twenty-four OTUs were shared between all RNA- and DNA-based libraries (OTU0.03) representing only 18% of the total OTUs, but 81% (1527/1883) of all sequences. Such differences between clone libraries demonstrate the necessity of generating both RNA- and DNA-derived clone libraries to compare these two different molecular approaches for community analyses.

  10. Improved protocol for the simultaneous extraction and column-based separation of DNA and RNA from different soils.

    PubMed

    Töwe, Stefanie; Wallisch, Stefanie; Bannert, Andrea; Fischer, Doreen; Hai, Brigitte; Haesler, Felix; Kleineidam, Kristina; Schloter, Michael

    2011-03-01

    We developed an improved protocol, allowing the simultaneous extraction of DNA and RNA from soil using phenol-chloroform with subsequent column-based separation of DNA and RNA (PCS). We compared this new approach with the well established protocol published by Griffiths et al. (2000), where DNA and RNA are separated by selective enzymatic digestions and two commercial kits used for DNA or RNA extraction, respectively, using four different agricultural soils. We compared yield and purity of the nucleic acids as well as abundance and diversity profiles of the soil bacterial communities targeting the nosZ gene via quantitative real-time PCR and terminal restriction fragment length polymorphism on DNA and RNA level. The newly developed protocol provided purer nucleic acid extracts compared to the used kit-based protocols. All protocols were suitable for DNA- and RNA-based gene quantification, however high variations between replicates were obtained for RNA samples using the original Griffiths protocol. Diversity patterns of nosZ were highly influenced by the extraction protocol used both on the DNA and RNA level. Finally, our data showed that the new protocol allows a simultaneous and reproducible extraction and separation of DNA and RNA, which were suitable for reliable analyses of gene and transcript copy numbers and diversity pattern. Copyright © 2011 Elsevier B.V. All rights reserved.

  11. HIV Viral RNA Extraction in Wax Immiscible Filtration Assisted by Surface Tension (IFAST) Devices

    PubMed Central

    Berry, Scott M.; LaVanway, Alex J.; Pezzi, Hannah M.; Guckenberger, David J.; Anderson, Meghan A.; Loeb, Jennifer M.; Beebe, David J.

    2015-01-01

    The monitoring of viral load is critical for proper management of antiretroviral therapy for HIV-positive patients. Unfortunately, in the developing world, significant economic and geographical barriers exist, limiting access to this test. The complexity of current viral load assays makes them expensive and their access limited to advanced facilities. We attempted to address these limitations by replacing conventional RNA extraction, one of the essential processes in viral load quantitation, with a simplified technique known as immiscible filtration assisted by surface tension (IFAST). Furthermore, these devices were produced via the embossing of wax, enabling local populations to produce and dispose of their own devices with minimal training or infrastructure, potentially reducing the total assay cost. In addition, IFAST can be used to reduce cold chain dependence during transportation. Viral RNA extracted from raw samples stored at 37°C for 1 week exhibited nearly complete degradation. However, IFAST-purified RNA could be stored at 37°C for 1 week without significant loss. These data suggest that RNA isolated at the point of care (eg, in a rural clinic) via IFAST could be shipped to a central laboratory for quantitative RT-PCR without a cold chain. Using this technology, we have demonstrated accurate and repeatable measurements of viral load on samples with as low as 50 copies per milliliter of sample. PMID:24613822

  12. Modified CTAB and TRIzol protocols improve RNA extraction from chemically complex Embryophyta1

    PubMed Central

    Jordon-Thaden, Ingrid E.; Chanderbali, Andre S.; Gitzendanner, Matthew A.; Soltis, Douglas E.

    2015-01-01

    Premise of the study: Here we present a series of protocols for RNA extraction across a diverse array of plants; we focus on woody, aromatic, aquatic, and other chemically complex taxa. Methods and Results: Ninety-one taxa were subjected to RNA extraction with three methods presented here: (1) TRIzol/TURBO DNA-free kits using the manufacturer’s protocol with the addition of sarkosyl; (2) a combination method using cetyltrimethylammonium bromide (CTAB) and TRIzol/sarkosyl/TURBO DNA-free; and (3) a combination of CTAB and QIAGEN RNeasy Plant Mini Kit. Bench-ready protocols are given. Conclusions: After an iterative process of working with chemically complex taxa, we conclude that the use of TRIzol supplemented with sarkosyl and the TURBO DNA-free kit is an effective, efficient, and robust method for obtaining RNA from 100 mg of leaf tissue of land plant species (Embryophyta) examined. Our protocols can be used to provide RNA of suitable stability, quantity, and quality for transcriptome sequencing. PMID:25995975

  13. Evaluation of RNA and DNA extraction from liquid-based cytology specimens.

    PubMed

    Fujii, Tomomi; Asano, Aya; Shimada, Keiji; Tatsumi, Yoshihiro; Obayashi, Chiho; Konishi, Noboru

    2016-10-01

    Molecular diagnosis using DNA and RNA derived from malignant tumors and molecular biological tools such as the quantitative polymerase-chain-reaction (qPCR) is a commonly used technique in clinical pathology. In this report, we compared the qualitative extraction of RNA and DNA from cancer cells fixed using several liquid-based cytology (LBC) kits. Ten to 1,000 cells from the T24 urinary bladder cancer cell line and SKG-II cervical cancer cell line were fixed with 55% methanol and three different methanol-based LBC solutions. The mRNA levels of CD44 in T24 cells and E7 in SKG-II cells and DNA levels of p53 in T24 cells and E7 in SKG-II cells were analyzed by qPCR. mRNA and DNA extracted from T24 and/or SKG-II cells fixed with methanol-based LBC solutions were efficiently detected, but to differing degrees, by qPCR. mRNA, and DNA from cells fixed with a formaldehyde-containing fixative liquid were detected at significantly low copy numbers by qPCR. Our results demonstrate that LBC systems are powerful tools for cytopathology and immunocytochemistry applications. However, the appropriate fixative must be selected for cell preservation when a small number of LBC samples is used for molecular testing, particularly in RNA-based molecular analyses. Diagn. Cytopathol. 2016;44:833-840. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  14. Automated solid-phase extraction coupled online with HPLC-FLD for the quantification of zearalenone in edible oil.

    PubMed

    Drzymala, Sarah S; Weiz, Stefan; Heinze, Julia; Marten, Silvia; Prinz, Carsten; Zimathies, Annett; Garbe, Leif-Alexander; Koch, Matthias

    2015-05-01

    Established maximum levels for the mycotoxin zearalenone (ZEN) in edible oil require monitoring by reliable analytical methods. Therefore, an automated SPE-HPLC online system based on dynamic covalent hydrazine chemistry has been developed. The SPE step comprises a reversible hydrazone formation by ZEN and a hydrazine moiety covalently attached to a solid phase. Seven hydrazine materials with different properties regarding the resin backbone, pore size, particle size, specific surface area, and loading have been evaluated. As a result, a hydrazine-functionalized silica gel was chosen. The final automated online method was validated and applied to the analysis of three maize germ oil samples including a provisionally certified reference material. Important performance criteria for the recovery (70-120 %) and precision (RSDr <25 %) as set by the Commission Regulation EC 401/2006 were fulfilled: The mean recovery was 78 % and RSDr did not exceed 8 %. The results of the SPE-HPLC online method were further compared to results obtained by liquid-liquid extraction with stable isotope dilution analysis LC-MS/MS and found to be in good agreement. The developed SPE-HPLC online system with fluorescence detection allows a reliable, accurate, and sensitive quantification (limit of quantification, 30 μg/kg) of ZEN in edible oils while significantly reducing the workload. To our knowledge, this is the first report on an automated SPE-HPLC method based on a covalent SPE approach.

  15. Efficient recovery of whole blood RNA--a comparison of commercial RNA extraction protocols for high-throughput applications in wildlife species.

    PubMed

    Schwochow, Doreen; Serieys, Laurel E K; Wayne, Robert K; Thalmann, Olaf

    2012-06-27

    Since the emergence of next generation sequencing platforms, unprecedented opportunities have arisen in the study of natural vertebrate populations. In particular, insights into the genetic and epigenetic mechanisms of adaptation can be revealed through study of the expression profiles of genes. However, as a pre-requisite to expression profiling, care must be taken in RNA preparation as factors like DNA contamination, RNA integrity or transcript abundance can affect downstream applications. Here, we evaluated five commonly used RNA extraction methods using whole blood sampled under varying conditions from 20 wild carnivores. Despite the use of minute starting volumes, all methods produced quantifiable RNA extracts (1.4 - 18.4 μg) with varying integrity (RIN 4.6 - 7.7), the latter being significantly affected by the storage and extraction method used. We observed a significant overall effect of the extraction method on DNA contamination. One particular extraction method, the LeukoLOCK™ filter system, yielded high RNA integrity along with low DNA contamination and efficient depletion of hemoglobin transcripts highly abundant in whole blood. In a proof of concept sequencing experiment, we found globin RNA transcripts to occupy up to ¼ of all sequencing reads if libraries were not depleted of hemoglobin prior to sequencing. By carefully choosing the appropriate RNA extraction method, whole blood can become a valuable source for high-throughput applications like expression arrays or transcriptome sequencing from natural populations. Additionally, candidate genes showing signs of selection could subsequently be genotyped in large population samples using whole blood as a source for RNA without harming individuals from rare or endangered species.

  16. Efficient recovery of whole blood RNA - a comparison of commercial RNA extraction protocols for high-throughput applications in wildlife species

    PubMed Central

    2012-01-01

    Background Since the emergence of next generation sequencing platforms, unprecedented opportunities have arisen in the study of natural vertebrate populations. In particular, insights into the genetic and epigenetic mechanisms of adaptation can be revealed through study of the expression profiles of genes. However, as a pre-requisite to expression profiling, care must be taken in RNA preparation as factors like DNA contamination, RNA integrity or transcript abundance can affect downstream applications. Here, we evaluated five commonly used RNA extraction methods using whole blood sampled under varying conditions from 20 wild carnivores. Results Despite the use of minute starting volumes, all methods produced quantifiable RNA extracts (1.4 – 18.4 μg) with varying integrity (RIN 4.6 - 7.7), the latter being significantly affected by the storage and extraction method used. We observed a significant overall effect of the extraction method on DNA contamination. One particular extraction method, the LeukoLOCK™ filter system, yielded high RNA integrity along with low DNA contamination and efficient depletion of hemoglobin transcripts highly abundant in whole blood. In a proof of concept sequencing experiment, we found globin RNA transcripts to occupy up to ¼ of all sequencing reads if libraries were not depleted of hemoglobin prior to sequencing. Conclusion By carefully choosing the appropriate RNA extraction method, whole blood can become a valuable source for high-throughput applications like expression arrays or transcriptome sequencing from natural populations. Additionally, candidate genes showing signs of selection could subsequently be genotyped in large population samples using whole blood as a source for RNA without harming individuals from rare or endangered species. PMID:22738215

  17. miRNA assays in the clinical laboratory: workflow, detection technologies and automation aspects.

    PubMed

    Kappel, Andreas; Keller, Andreas

    2017-05-01

    microRNAs (miRNAs) are short non-coding RNA molecules that regulate gene expression in eukaryotes. Their differential abundance is indicative or even causative for a variety of pathological processes including cancer or cardiovascular disorders. Due to their important biological function, miRNAs represent a promising class of novel biomarkers that may be used to diagnose life-threatening diseases, and to monitor disease progression. Further, they may guide treatment selection or dosage of drugs. miRNAs from blood or derived fractions are particularly interesting candidates for routine laboratory applications, as they can be measured in most clinical laboratories already today. This assures a good accessibility of respective tests. Albeit their great potential, miRNA-based diagnostic tests have not made their way yet into the clinical routine, and hence no standardized workflows have been established to measure miRNAs for patients' benefit. In this review we summarize the detection technologies and workflow options that exist to measure miRNAs, and we describe the advantages and disadvantages of each of these options. Moreover, we also provide a perspective on data analysis aspects that are vital for translation of raw data into actionable diagnostic test results.

  18. A highly efficient method for extracting next-generation sequencing quality RNA from adipose tissue of recalcitrant animal species.

    PubMed

    Sharma, Davinder; Golla, Naresh; Singh, Dheer; Onteru, Suneel Kumar

    2017-04-13

    The next-generation sequencing (NGS) based RNA sequencing (RNA-Seq) and transcriptome profiling offers an opportunity to unveil complex evolutionary processes. Successful RNA-Seq and transcriptome profiling requires a large amount of high-quality RNA. However, NGS-quality RNA isolation is extremely difficult from recalcitrant adipose tissue (AT) with high lipid content and low cell numbers. Further, the amount and biochemical composition of AT lipid varies depending upon the animal species which can pose different degree of resistance to RNA extraction. Currently available approaches may work effectively in one species but can be almost unproductive in another species. Herein, we report a two step protocol for the extraction of NGS quality RNA from AT across a broad range of animal species. This article is protected by copyright. All rights reserved.

  19. Preparation of Formalin-fixed Paraffin-embedded Tissue Cores for both RNA and DNA Extraction.

    PubMed

    Patel, Palak G; Selvarajah, Shamini; Boursalie, Suzanne; How, Nathan E; Ejdelman, Joshua; Guerard, Karl-Philippe; Bartlett, John M; Lapointe, Jacques; Park, Paul C; Okello, John B A; Berman, David M

    2016-08-21

    Formalin-fixed paraffin embedded tissue (FFPET) represents a valuable, well-annotated substrate for molecular investigations. The utility of FFPET in molecular analysis is complicated both by heterogeneous tissue composition and low yields when extracting nucleic acids. A literature search revealed a paucity of protocols addressing these issues, and none that showed a validated method for simultaneous extraction of RNA and DNA from regions of interest in FFPET. This method addresses both issues. Tissue specificity was achieved by mapping cancer areas of interest on microscope slides and transferring annotations onto FFPET blocks. Tissue cores were harvested from areas of interest using 0.6 mm microarray punches. Nucleic acid extraction was performed using a commercial FFPET extraction system, with modifications to homogenization, deparaffinization, and Proteinase K digestion steps to improve tissue digestion and increase nucleic acid yields. The modified protocol yields sufficient quantity and quality of nucleic acids for use in a number of downstream analyses, including a multi-analyte gene expression platform, as well as reverse transcriptase coupled real time PCR analysis of mRNA expression, and methylation-specific PCR (MSP) analysis of DNA methylation.

  20. Automated Feature Extraction by Combining Polarimetric SAR and Object-Based Image Analysis for Monitoring of Natural Resource Exploitation

    NASA Astrophysics Data System (ADS)

    Plank, Simon; Mager, Alexander; Schoepfer, Elizabeth

    2015-04-01

    An automated feature extraction procedure based on the combination of a pixel-based unsupervised classification of polarimetric synthetic aperture radar data (co-co dual-polarimetric TerraSAR-X) and an object-based post-classification is presented. The former is based on the entropy/alpha decomposition and the hereon based unsupervised Wishart classification, while the latter considers in addition feature properties such as shape and area. The feature extraction procedure is developed for monitoring oil field infrastructure. For developing countries, several studies reported a high correlation between the dependence of oil exports and violent conflicts. Consequently, to support problem solving, an independent monitoring of the oil field infrastructure by Earth observation is proposed.

  1. Extraction platform evaluations: a comparison of AutoMate Express™, EZ1® Advanced XL, and Maxwell® 16 Bench-top DNA extraction systems.

    PubMed

    Davis, Carey P; King, Jonathan L; Budowle, Bruce; Eisenberg, Arthur J; Turnbough, Meredith A

    2012-01-01

    The DNA extraction performance of three low-throughput extraction systems was evaluated. The instruments and respective chemistries all use a similar extraction methodology that involves binding DNA to a coated magnetic resin in the presence of chaotropic salt, washing of the resin to remove undesirable compounds, and elution of DNA from the particles in a low-salt solution. The AutoMate Express™ (Life Technologies Corporation, Carlsbad, CA), EZ1® Advanced XL (Qiagen Inc., Valencia, CA), and Maxwell® 16 (Promega Corporation, Madison, WI) were compared using a variety of samples including: blood on swabs, blood on denim, blood on cotton, blood mixed with inhibitors (a mixture of indigo, hematin, humic acid, and urban dust) on cotton, blood on FTA® paper, saliva residue on cigarette butt paper, epithelial cells on cotton swabs, neat semen on cotton, hair roots, bones, and teeth. Each instrument had a recommended pre-processing protocol for each sample type, and these protocols were followed strictly to reduce user bias. All extractions were performed in triplicate for each sample type. The three instruments were compared on the basis of quantity of DNA recovered (as determined by real-time PCR), relative level of inhibitors present in the extract (shown as shifts in the C(T) value for the internal PCR control in the real-time PCR assay), STR peak heights, use of consumables not included in the extraction kits, ease of use, and application flexibility. All three systems performed well; however extraction efficiency varied by sample type and with the preprocessing protocol applied to the various samples. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

  2. Soil DNA Extraction Procedure Influences Protist 18S rRNA Gene Community Profiling Outcome.

    PubMed

    Santos, Susana S; Nunes, Inês; Nielsen, Tue K; Jacquiod, Samuel; Hansen, Lars H; Winding, Anne

    2017-07-01

    Advances in sequencing technologies allow deeper studies of the soil protist diversity and function. However, little attention has been given to the impact of the chosen soil DNA extraction procedure to the overall results. We examined the effect of three acknowledged DNA recovery methods, two manual methods (ISOm-11063, GnS-GII) and one commercial kit (MoBio), on soil protist community structures obtained from different sites with different land uses. Results from 18S rRNA gene amplicon sequencing suggest that DNA extraction method significantly affect the replicate homogeneity, the total number of operational taxonomic units (OTUs) recovered and the overall taxonomic structure and diversity of soil protist communities. However, DNA extraction effects did not overwhelm the natural variation among samples, as the community data still strongly grouped by geographical location. The commercial DNA extraction kit was associated with the highest diversity estimates and with a corresponding higher retrieval of Excavata, Cercozoa and Amoebozoa-related taxa. Overall, our findings indicate that this extraction offers a compromise between rare and dominant taxa representation, while providing high replication reproducibility. A comprehensive understanding of the DNA extraction techniques impact on soil protist diversity can enable more accurate diversity assays. Copyright © 2017 Elsevier GmbH. All rights reserved.

  3. Novel automated extraction method for quantitative analysis of urinary 11-nor-delta(9)-tetrahydrocannabinol-9-carboxylic acid (THC-COOH).

    PubMed

    Fu, Shanlin; Lewis, John

    2008-05-01

    An automated extraction method for extracting the major urinary metabolite of cannabis, 11-nor-Delta(9)-tetrahydrocannabinol-9-carboxylic acid (THC-COOH) was developed on the four-probe Gilson ASPEC XL4trade mark solid-phase extraction (SPE) system. The method works on liquid-liquid extraction principles but does not require the use of SPE cartridges. The limits of detection and quantitation and the upper limit of linearity (ULOL) of the developed method were found to be 1, 2, and 1,500 ng/mL, respectively. There was no detectable carry over after 10,000 ng/mL analyte. For a batch of 76 samples, the process uses less than 100 mL methanol, 450 mL extracting solvent hexane/ethyl acetate (5:1, v/v) and 1 L rinsing solvent, 30% methanol in water. The automated extraction process takes 5 h to complete. Precision and accuracy of the method are comparable to both manual liquid-liquid extraction and automated SPE methods. The method has proven to be a simple, speedy, and economical alternative to the currently popular automated SPE method for the quantitative analysis of urinary THC-COOH.

  4. Viral RNA testing and automation on the bead-based CBNE detection microsystem.

    SciTech Connect

    Galambos, Paul C.; Bourdon, Christopher Jay; Farrell, Cara M.; Rossito, Paul; McClain, Jaime L.; Derzon, Mark Steven; Cullor, James Sterling; Rahimian, Kamayar

    2008-09-01

    We developed prototype chemistry for nucleic acid hybridization on our bead-based diagnostics platform and we established an automatable bead handling protocol capable of 50 part-per-billion (ppb) sensitivity. We are working towards a platform capable of parallel, rapid (10 minute), raw sample testing for orthogonal (in this case nucleic acid and immunoassays) identification of biological (and other) threats in a single sensor microsystem. In this LDRD we developed the nucleic acid chemistry required for nucleic acid hybridization. Our goal is to place a non-cell associated RNA virus (Bovine Viral Diarrhea, BVD) on the beads for raw sample testing. This key pre-requisite to showing orthogonality (nucleic acid measurements can be performed in parallel with immunoassay measurements). Orthogonal detection dramatically reduces false positives. We chose BVD because our collaborators (UC-Davis) can supply samples from persistently infected animals; and because proof-of-concept field testing can be performed with modification of the current technology platform at the UC Davis research station. Since BVD is a cattle-prone disease this research dovetails with earlier immunoassay work on Botulinum toxin simulant testing in raw milk samples. Demonstration of BVD RNA detection expands the repertoire of biological macromolecules that can be adapted to our bead-based detection. The resources of this late start LDRD were adequate to partially demonstrate the conjugation of the beads to the nucleic acids. It was never expected to be adequate for a full live virus test but to motivate that additional investment. In addition, we were able to reduce the LOD (Limit of Detection) for the botulinum toxin stimulant to 50 ppb from the earlier LOD of 1 ppm. A low LOD combined with orthogonal detection provides both low false negatives and low false positives. The logical follow-on steps to this LDRD research are to perform live virus identification as well as concurrent nucleic acid and

  5. Screening for anabolic steroids in urine of forensic cases using fully automated solid phase extraction and LC-MS-MS.

    PubMed

    Andersen, David W; Linnet, Kristian

    2014-01-01

    A screening method for 18 frequently measured exogenous anabolic steroids and the testosterone/epitestosterone (T/E) ratio in forensic cases has been developed and validated. The method involves a fully automated sample preparation including enzyme treatment, addition of internal standards and solid phase extraction followed by analysis by liquid chromatography-tandem mass spectrometry (LC-MS-MS) using electrospray ionization with adduct formation for two compounds. Urine samples from 580 forensic cases were analyzed to determine the T/E ratio and occurrence of exogenous anabolic steroids. Extraction recoveries ranged from 77 to 95%, matrix effects from 48 to 78%, overall process efficiencies from 40 to 54% and the lower limit of identification ranged from 2 to 40 ng/mL. In the 580 urine samples analyzed from routine forensic cases, 17 (2.9%) were found positive for one or more anabolic steroids. Only seven different steroids including testosterone were found in the material, suggesting that only a small number of common steroids are likely to occur in a forensic context. The steroids were often in high concentrations (>100 ng/mL), and a combination of steroids and/or other drugs of abuse were seen in the majority of cases. The method presented serves as a fast and automated screening procedure, proving the suitability of LC-MS-MS for analyzing anabolic steroids. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  6. Control of 5S RNA transcription in Xenopus somatic cell chromatin: activation with an oocyte extract.

    PubMed Central

    Reynolds, W F; Bloomer, L S; Gottesfeld, J M

    1983-01-01

    A chromatin fraction enriched for Xenopus 5S RNA genes has been isolated by restriction endonuclease digestion and sucrose gradient velocity sedimentation. Soluble chromatin sedimenting at 70-80S contains approximately 50% of the oocyte-expressed 5S RNA genes and only 1.5-3% of total chromatin DNA; this represents a 15- to 30-fold purification of the 5S genes. Such chromatin isolated from somatic cells (blood and cultured kidney cells) retains the transcriptionally-inactive state of the oocyte-expressed 5S genes. Soluble chromatin from somatic cells prepared by micrococcal nuclease digestion also retains the inactive state of the oocyte-type 5S genes. It is likely that the level of chromatin structure responsible for inactivity of the oocyte genes in somatic cells is the nucleosome or short chains of nucleosomes and not supranucleosomal structures. The oocyte-type genes can be rendered transcriptionally active in somatic cell chromatin either by salt extraction of some chromosomal proteins or by treatment with the ion exchange resin Dowex A50W-X2. Alternatively, activation of these genes can be achieved by incubating somatic cell chromatin or nuclei with an extract prepared from Xenopus oocytes. This effect is not specific for 5S RNA genes as the transcription of other small RNAs (including pre-tRNA) is stimulated by the oocyte extract. The activating factor(s) is resistant to micrococcal nuclease, nondialyzable, heat labile and sensitive to trypsin; thus it is highly likely to be a protein or a group of proteins. Partial purification of the activating factor(s) has been achieved by ion exchange chromatography. Images PMID:6866764

  7. INVESTIGATION OF ARSENIC SPECIATION ON DRINKING WATER TREATMENT MEDIA UTILIZING AUTOMATED SEQUENTIAL CONTINUOUS FLOW EXTRACTION WITH IC-ICP-MS DETECTION

    EPA Science Inventory

    Three treatment media, used for the removal of arsenic from drinking water, were sequentially extracted using 10mM MgCl2 (pH 8), 10mM NaH2PO4 (pH 7) followed by 10mM (NH4)2C2O4 (pH 3). The media were extracted using an on-line automated continuous extraction system which allowed...

  8. INVESTIGATION OF ARSENIC SPECIATION ON DRINKING WATER TREATMENT MEDIA UTILIZING AUTOMATED SEQUENTIAL CONTINUOUS FLOW EXTRACTION WITH IC-ICP-MS DETECTION

    EPA Science Inventory

    Three treatment media, used for the removal of arsenic from drinking water, were sequentially extracted using 10mM MgCl2 (pH 8), 10mM NaH2PO4 (pH 7) followed by 10mM (NH4)2C2O4 (pH 3). The media were extracted using an on-line automated continuous extraction system which allowed...

  9. Direct Sampling and Analysis from Solid Phase Extraction Cards using an Automated Liquid Extraction Surface Analysis Nanoelectrospray Mass Spectrometry System

    SciTech Connect

    Walworth, Matthew J; ElNaggar, Mariam S; Stankovich, Joseph J; WitkowskiII, Charles E.; Norris, Jeremy L; Van Berkel, Gary J

    2011-01-01

    Direct liquid extraction based surface sampling, a technique previously demonstrated with continuous flow and autonomous pipette liquid microjunction surface sampling probes, has recently been implemented as the Liquid Extraction Surface Analysis (LESA) mode on the commercially available Advion NanoMate chip-based infusion nanoelectrospray ionization system. In the present paper, the LESA mode was applied to the analysis of 96-well format custom solid phase extraction (SPE) cards, with each well consisting of either a 1 or 2 mm diameter monolithic hydrophobic stationary phase. These substrate wells were conditioned, loaded with either single or multi-component aqueous mixtures, and read out using the LESA mode of a TriVersa NanoMate or a Nanomate 100 coupled to an ABI/Sciex 4000QTRAPTM hybrid triple quadrupole/linear ion trap mass spectrometer and a Thermo LTQ XL linear ion trap mass spectrometer. Extraction conditions, including extraction/nanoESI solvent composition, volume, and dwell times, were optimized in the analysis of targeted compounds. Limit of detection and quantitation as well as analysis reproducibility figures of merit were measured. Calibration data was obtained for propranolol using a deuterated internal standard which demonstrated linearity and reproducibility. A 10x increase in signal and cleanup of micromolar Angiotensin II from a concentrated salt solution was demonstrated. Additionally, a multicomponent herbicide mixture at ppb concentration levels was analyzed using MS3 spectra for compound identification in the presence of isobaric interferences.

  10. A method for DNA and RNA co-extraction for use on forensic samples using the Promega DNA IQ™ system.

    PubMed

    Bowden, Anna; Fleming, Rachel; Harbison, SallyAnn

    2011-01-01

    The use of messenger RNA profiling to identify the origin of biological samples (e.g. blood, semen and saliva) from crime scenes is now at the stage of being implemented into routine forensic casework. We report on the successful modification of the Promega DNA IQ™ system to enable co-extraction of DNA and RNA from the same sample without compromising the potential DNA profile. Using the protocol in our laboratory for extracting DNA using the DNA IQ™ system combined with the Zymo Research Mini RNA Isolation Kit™ II we demonstrate the simultaneous co-extraction of DNA and RNA from the same sample for routine DNA and mRNA profiling for the identification of both the individual and the biological stain.

  11. Olive Leaf Extract Elevates Hepatic PPAR α mRNA Expression and Improves Serum Lipid Profiles in Ovariectomized Rats.

    PubMed

    Yoon, Leena; Liu, Ya-Nan; Park, Hyunjin; Kim, Hyun-Sook

    2015-07-01

    We hypothesized that olive leaf extract might alleviate dyslipidemia resulting from estrogen deficiency. Serum lipid profile and mRNA expression of the related genes in the liver and adipose tissue were analyzed after providing olive leaf extract (200 or 400 mg/kg body weight; n=7 for each group) to ovariectomized rats for 10 weeks. After 10 weeks' administration, the rats in the olive leaf extract-administered groups showed significantly lower levels of serum triglyceride and very-low-density lipoprotein (VLDL)-cholesterol compared with the rats in the control group, whereas the administration of olive leaf extract did not significantly change the elevated low-density lipoprotein cholesterol levels. In addition, administration of high dose of olive leaf extract significantly decreased the liver triglyceride and increased serum estradiol levels. mRNA expressions of peroxisome proliferator-activated receptor alpha (PPAR α) and acyl-CoA oxidase (ACO) were not affected by ovariectomy, however, administration of olive leaf extract significantly increased both PPAR α and ACO mRNA expression. Expression of adiponectin mRNA in adipose tissue was significantly decreased in the ovariectomized control group. Rats administered low-dose olive leaf extract showed significantly elevated adiponectin mRNA expression compared with rats in the ovariectomized control group. Even though dose-dependent effects were not observed in most of the measurements, these results suggest that genes involved in lipid metabolism may be regulated by olive leaf extract administration in ovariectomized rats.

  12. [The extract of compound Radix Scutellariae on mRNA replication and IFN expression of influenza virus in mice].

    PubMed

    Chu, Ming; Chu, Zheng-yun; Wang, Dan-dan

    2007-01-01

    To study the extract of compound Radix Scutellariae on inhibiting the mRNA replication and inducing the INF expression of influenza virus in mice's vivo. Influenza virus infected mice pneumono-adaption stock A/FM/1/47 (H1N1) evoked mice pneumonia taken as animal model. RT-PCR was adopted to measure the content of influenza virus's mRNA and INF in mice to study the effect of the extract on mRNA replication and IFN expression. The extract of compound Radix Scutellariae could inhibit influenza virus' s mRNA replication (P < 0.01) and induce interferon expression in mice (P > 0.05). The extract of compound Radix Scutellariae can induce interferon and inhibit influenza virus replication in mice.

  13. Automated wide-angle SAR stereo height extraction in rugged terrain using shift-scaling correlation.

    SciTech Connect

    Yocky, David Alan; Jakowatz, Charles V., Jr.

    2003-07-01

    Coherent stereo pairs from cross-track synthetic aperture radar (SAR) collects allow fully automated correlation matching using magnitude and phase data. Yet, automated feature matching (correspondence) becomes more difficult when imaging rugged terrain utilizing large stereo crossing angle geometries because high-relief features can undergo significant spatial distortions. These distortions sometimes cause traditional, shift-only correlation matching to fail. This paper presents a possible solution addressing this difficulty. Changing the complex correlation maximization search from shift-only to shift-and-scaling using the downhill simplex method results in higher correlation. This is shown on eight coherent spotlight-mode cross-track stereo pairs with stereo crossing angles averaging 93.7{sup o} collected over terrain with slopes greater than 20{sup o}. The resulting digital elevation maps (DEMs) are compared to ground truth. Using the shift-scaling correlation approach to calculate disparity, height errors decrease and the number of reliable DEM posts increase.

  14. Sequential Chomospheric Brightening: An Automated Approach to Extracting Physics from Ephemeral Brightening

    DTIC Science & Technology

    2012-10-17

    University; 2 Space Vehicles Directorate, Air Force Research Laboratory; 3 National Solar Observatory, Sunspot, NM; 4 Astrophysics Research Centre...propose a connection of the small-scale features to solar flares. Our automated routine detects and distinguishes three separate types of brightening...88003-8001; mskirk@nmsu.edu 2 Space Vehicles Directorate, Air Force Research Laboratory, Kirtland AFB, NM 87114 3 National Solar Observatory, Sunspot

  15. High performance liquid chromatography for quantification of gatifloxacin in rat plasma following automated on-line solid phase extraction.

    PubMed

    Tasso, Leandro; Dalla Costa, Teresa

    2007-05-09

    An automated system using on-line solid phase extraction and HPLC with fluorimetric detection was developed and validated for quantification of gatifloxacin in rat plasma. The extraction was carried out using C(18) cartridges (BondElut), with a high extraction yield. After washing, gatifloxacin was eluted from the cartridge with mobile phase onto a C(18) HPLC column. The mobile phase consisted of a mixture of phosphoric acid (2.5mM), methanol, acetonitrile and triethylamine (64.8:15:20:0.2, v/v/v/v, apparent pH(app.) 2.8). All samples and standard solutions were chromatographed at 28 degrees C. The method developed was selective and linear for drug concentrations ranging between 20 and 600 ng/ml. Gatifloxacin recovery ranged from 95.6 to 99.7%, and the limit of quantification was 20 ng/ml. The intra and inter-assay accuracy were up to 94.3%. The precision determined not exceed 5.8% of the CV. High extraction yield up to 95% was obtained. Drug stability in plasma was shown in freezer at -20 degrees C up to 1 month, after three freeze-thaw cycles and for 24h in the autosampler after processing. The assay has been successfully applied to measure gatifloxacin plasma concentrations in pharmacokinetic study in rats.

  16. Performance verification of the Maxwell 16 Instrument and DNA IQ Reference Sample Kit for automated DNA extraction of known reference samples.

    PubMed

    Krnajski, Z; Geering, S; Steadman, S

    2007-12-01

    Advances in automation have been made for a number of processes conducted in the forensic DNA laboratory. However, because most robotic systems are designed for high-throughput laboratories batching large numbers of samples, smaller laboratories are left with a limited number of cost-effective options for employing automation. The Maxwell 16 Instrument and DNA IQ Reference Sample Kit marketed by Promega are designed for rapid, automated purification of DNA extracts from sample sets consisting of sixteen or fewer samples. Because the system is based on DNA capture by paramagnetic particles with maximum binding capacity, it is designed to generate extracts with yield consistency. The studies herein enabled evaluation of STR profile concordance, consistency of yield, and cross-contamination performance for the Maxwell 16 Instrument. Results indicate that the system performs suitably for streamlining the process of extracting known reference samples generally used for forensic DNA analysis and has many advantages in a small or moderate-sized laboratory environment.

  17. Semi-automated solid-phase extraction method for studying the biodegradation of ochratoxin A by human intestinal microbiota.

    PubMed

    Camel, Valérie; Ouethrani, Minale; Coudray, Cindy; Philippe, Catherine; Rabot, Sylvie

    2012-04-15

    A simple and rapid semi-automated solid-phase (SPE) extraction method has been developed for the analysis of ochratoxin A in aqueous matrices related to biodegradation experiments (namely digestive contents and faecal excreta), with a view of using this method to follow OTA biodegradation by human intestinal microbiota. Influence of extraction parameters that could affect semi-automated SPE efficiency was studied, using C18-silica as the sorbent and water as the simplest matrix, being further applied to the matrices of interest. Conditions finally retained were as follows: 5-mL aqueous samples (pH 3) containing an organic modifier (20% ACN) were applied on 100-mg cartridges. After drying (9 mL of air), the cartridge was rinsed with 5-mL H(2)O/ACN (80:20, v/v), before eluting the compounds with 3 × 1 mL of MeOH/THF (10:90, v/v). Acceptable recoveries and limits of quantification could be obtained considering the complexity of the investigated matrices and the low volumes sampled; this method was also suitable for the analysis of ochratoxin B in faecal extracts. Applicability of the method is illustrated by preliminary results of ochratoxin A biodegradation studies by human intestinal microbiota under simple in vitro conditions. Interestingly, partial degradation of ochratoxin A was observed, with efficiencies ranging from 14% to 47% after 72 h incubation. In addition, three phase I metabolites could be identified using high resolution mass spectrometry, namely ochratoxin α, open ochratoxin A and ochratoxin B.

  18. A sequential co-extraction method for DNA, RNA and protein recovery from soil for future system-based approaches.

    PubMed

    Gunnigle, Eoin; Ramond, Jean-Baptiste; Frossard, Aline; Seeley, Mary; Cowan, Don

    2014-08-01

    A co-extraction protocol that sequentially isolates core biopolymer fractions (DNA, RNA, protein) from edaphic microbial communities is presented. In order to confirm compatibility with downstream analyses, bacterial T-RFLP profiles were generated from the DNA- and RNA-derived fractions of an arid-based soil, with metaproteomics undertaken on the corresponding protein fraction.

  19. Automated on-line liquid-liquid extraction system for temporal mass spectrometric analysis of dynamic samples.

    PubMed

    Hsieh, Kai-Ta; Liu, Pei-Han; Urban, Pawel L

    2015-09-24

    Most real samples cannot directly be infused to mass spectrometers because they could contaminate delicate parts of ion source and guides, or cause ion suppression. Conventional sample preparation procedures limit temporal resolution of analysis. We have developed an automated liquid-liquid extraction system that enables unsupervised repetitive treatment of dynamic samples and instantaneous analysis by mass spectrometry (MS). It incorporates inexpensive open-source microcontroller boards (Arduino and Netduino) to guide the extraction and analysis process. Duration of every extraction cycle is 17 min. The system enables monitoring of dynamic processes over many hours. The extracts are automatically transferred to the ion source incorporating a Venturi pump. Operation of the device has been characterized (repeatability, RSD = 15%, n = 20; concentration range for ibuprofen, 0.053-2.000 mM; LOD for ibuprofen, ∼0.005 mM; including extraction and detection). To exemplify its usefulness in real-world applications, we implemented this device in chemical profiling of pharmaceutical formulation dissolution process. Temporal dissolution profiles of commercial ibuprofen and acetaminophen tablets were recorded during 10 h. The extraction-MS datasets were fitted with exponential functions to characterize the rates of release of the main and auxiliary ingredients (e.g. ibuprofen, k = 0.43 ± 0.01 h(-1)). The electronic control unit of this system interacts with the operator via touch screen, internet, voice, and short text messages sent to the mobile phone, which is helpful when launching long-term (e.g. overnight) measurements. Due to these interactive features, the platform brings the concept of the Internet-of-Things (IoT) to the chemistry laboratory environment. Copyright © 2015 Elsevier B.V. All rights reserved.

  20. A high yield DNA extraction method for medically important Candida species: A comparison of manual versus QIAcube-based automated system.

    PubMed

    Das, P; Pandey, P; Harishankar, A; Chandy, M; Bhattacharya, S

    2016-01-01

    The prognosis of infected individuals with candidemia depends on rapid and precise diagnosis which enables optimising treatment. Three fungal DNA extraction protocols have been compared in this study for medically important Candida species. The quality and quantity of the DNA extracted by physical, chemical and automated protocols was compared using NanoDrop ND-2000 spectrophotometer. It was found that the yield and purity (260/230) ratio of extracted DNA was significantly high in the physical treatment-based protocol as compared to chemical based or automated protocol. Extracted DNA-based real time-polymerase chain reaction showed an analytical sensitivity of 103 cfu/mL. The result of this study suggests physical treatment is the most successful extraction technique compared to other two protocols.

  1. [Efficiency of three commercial kits dedicated to DNA and RNA isolation from various clinical and forensic materials using the Janus automated workstation].

    PubMed

    Małodobra, Małgorzata; Jonkisz, Anna; Kowalczyk, Elzbieta; Lebioda, Arleta; Bartnik, Beata; Swiatek, Barbara

    2011-01-01

    Isolation of genetic material is a crucial stage in molecular biology. Increasing needs for DNA analysis cause continuous improving of genetic material isolation methods toward higher accuracy and output. Automatization in molecular biology is widely seen, especially in clinical and forensic medicine. The objective of this research was optimization of methods for automatic nucleic acid isolation using the Janus automated workstation, Perkin Elmer. The efficiency and purity of isolated DNA was satisfactory. Despite numerous attempts at achieving automatic RNA isolation, we did not succeed in obtaining RNA working in other applications, such as RT-PCR or Real-Time PCR.

  2. Automated methods of tree boundary extraction and foliage transparency estimation from digital imagery

    Treesearch

    Sang-Mook Lee; Neil A. Clark; Philip A. Araman

    2003-01-01

    Foliage transparency in trees is an important indicator for forest health assessment. This paper helps advance transparency measurement research by presenting methods of automatic tree boundary extraction and foliage transparency estimation from digital images taken from the ground of open grown trees.Extraction of proper boundaries of tree crowns is the...

  3. Membrane-assisted culture of fungal mycelium on agar plates for RNA extraction and pharmacological analyses.

    PubMed

    Lange, Mario; Müller, Carolin; Peiter, Edgar

    2014-05-15

    Fungal mycelium grown in liquid culture is easy to harvest for RNA extraction and gene expression analyses, but liquid cultures often develop rather heterogeneously. In contrast, growth of fungal mycelium on agar plates is highly reproducible. However, this biological material cannot be harvested easily for downstream analyses. This article describes a PVDF (polyvinylidene difluoride) membrane-assisted agar plate culture method that enables the harvest of mycelium grown on agar plates. This culture method leads to a strongly reduced variation in gene expression between biological replicates and requires less growth space as compared with liquid cultures.

  4. [Influence of temperature on the preferential extraction of RNA polymerase I from hepatic nuclei of the rat].

    PubMed

    Zoncheddu, A; Accomando, R; Pertica, M; Orunesu, M

    1979-11-15

    RNA polymerase I has been extracted from rat liver nuclei by three consecutive washings at 0 degrees C with a medium of relatively low ionic strength (0.15 M KCl) containing Mg++ rather than by incubating the organelles at 37 degrees C in the same medium, as originally proposed by Chesterton and Butterworth. The modified technique, which has the advantage of preventing a temperature-mediated conversion of form IB to IA, gives similar yields of RNA polymerase I and retains the capacity of preferentially extracting the enzyme with respect to the other forms of nuclear RNA polymerase.

  5. Characterization and Application of Superlig 620 Solid Phase Extraction Resin for Automated Process Monitoring of 90Sr

    SciTech Connect

    Devol, Timothy A.; Clements, John P.; Farawila, Anne F.; O'Hara, Matthew J.; Egorov, Oleg; Grate, Jay W.

    2009-11-30

    Characterization of SuperLig® 620 solid phase extraction resin was performed in order to develop an automated on-line process monitor for 90Sr. The main focus was on strontium separation from barium, with the goal of developing an automated separation process for 90Sr in high-level wastes. High-level waste contains significant 137Cs activity, of which 137mBa is of great concern as an interference to the quantification of strontium. In addition barium, yttrium and plutonium were studied as potential interferences to strontium uptake and detection. A number of complexants were studied in a series of batch Kd experiments, as SuperLig® 620 was not previously known to elute strontium in typical mineral acids. The optimal separation was found using a 2M nitric acid load solution with a strontium elution step of ~0.49M ammonium citrate and a barium elution step of ~1.8M ammonium citrate. 90Sr quantification of Hanford high-level tank waste was performed on a sequential injection analysis microfluidics system coupled to a flow-cell detector. The results of the on-line procedure are compared to standard radiochemical techniques in this paper.

  6. Automated Control of the Organic and Inorganic Composition of Aloe vera Extracts Using (1)H NMR Spectroscopy.

    PubMed

    Monakhova, Yulia B; Randel, Gabriele; Diehl, Bernd W K

    2016-09-01

    Recent classification of Aloe vera whole-leaf extract by the International Agency for Research and Cancer as a possible carcinogen to humans as well as the continuous adulteration of A. vera's authentic material have generated renewed interest in controlling A. vera. The existing NMR spectroscopic method for the analysis of A. vera, which is based on a routine developed at Spectral Service, was extended. Apart from aloverose, glucose, malic acid, lactic acid, citric acid, whole-leaf material (WLM), acetic acid, fumaric acid, sodium benzoate, and potassium sorbate, the quantification of Mg(2+), Ca(2+), and fructose is possible with the addition of a Cs-EDTA solution to sample. The proposed methodology was automated, which includes phasing, baseline-correction, deconvolution (based on the Lorentzian function), integration, quantification, and reporting. The NMR method was applied to 41 A. vera preparations in the form of liquid A. vera juice and solid A. vera powder. The advantages of the new NMR methodology over the previous method were discussed. Correlation between the new and standard NMR methodologies was significant for aloverose, glucose, malic acid, lactic acid, citric acid, and WLM (P < 0.0001, R(2) = 0.99). NMR was found to be suitable for the automated simultaneous quantitative determination of 13 parameters in A. vera.

  7. Feature extraction from terahertz pulses for classification of RNA data via support vector machines

    NASA Astrophysics Data System (ADS)

    Yin, Xiaoxia; Ng, Brian W.-H.; Fischer, Bernd; Ferguson, Bradley; Mickan, Samuel P.; Abbott, Derek

    2006-12-01

    This study investigates binary and multiple classes of classification via support vector machines (SVMs). A couple of groups of two dimensional features are extracted via frequency orientation components, which result in the effective classification of Terahertz (T-ray) pulses for discrimination of RNA data and various powder samples. For each classification task, a pair of extracted feature vectors from the terahertz signals corresponding to each class is viewed as two coordinates and plotted in the same coordinate system. The current classification method extracts specific features from the Fourier spectrum, without applying an extra feature extractor. This method shows that SVMs can employ conventional feature extraction methods for a T-ray classification task. Moreover, we discuss the challenges faced by this method. A pairwise classification method is applied for the multi-class classification of powder samples. Plots of learning vectors assist in understanding the classification task, which exhibit improved clustering, clear learning margins, and least support vectors. This paper highlights the ability to use a small number of features (2D features) for classification via analyzing the frequency spectrum, which greatly reduces the computation complexity in achieving the preferred classification performance.

  8. Comparison of Boiling and Robotics Automation Method in DNA Extraction for Metagenomic Sequencing of Human Oral Microbes

    PubMed Central

    Shinozaki, Natsuko; Ye, Bin; Tsuboi, Akito; Nagasaki, Masao; Yamashita, Riu

    2016-01-01

    The rapid improvement of next-generation sequencing performance now enables us to analyze huge sample sets with more than ten thousand specimens. However, DNA extraction can still be a limiting step in such metagenomic approaches. In this study, we analyzed human oral microbes to compare the performance of three DNA extraction methods: PowerSoil (a method widely used in this field), QIAsymphony (a robotics method), and a simple boiling method. Dental plaque was initially collected from three volunteers in the pilot study and then expanded to 12 volunteers in the follow-up study. Bacterial flora was estimated by sequencing the V4 region of 16S rRNA following species-level profiling. Our results indicate that the efficiency of PowerSoil and QIAsymphony was comparable to the boiling method. Therefore, the boiling method may be a promising alternative because of its simplicity, cost effectiveness, and short handling time. Moreover, this method was reliable for estimating bacterial species and could be used in the future to examine the correlation between oral flora and health status. Despite this, differences in the efficiency of DNA extraction for various bacterial species were observed among the three methods. Based on these findings, there is no “gold standard” for DNA extraction. In future, we suggest that the DNA extraction method should be selected on a case-by-case basis considering the aims and specimens of the study. PMID:27104353

  9. Comparison of Boiling and Robotics Automation Method in DNA Extraction for Metagenomic Sequencing of Human Oral Microbes.

    PubMed

    Yamagishi, Junya; Sato, Yukuto; Shinozaki, Natsuko; Ye, Bin; Tsuboi, Akito; Nagasaki, Masao; Yamashita, Riu

    2016-01-01

    The rapid improvement of next-generation sequencing performance now enables us to analyze huge sample sets with more than ten thousand specimens. However, DNA extraction can still be a limiting step in such metagenomic approaches. In this study, we analyzed human oral microbes to compare the performance of three DNA extraction methods: PowerSoil (a method widely used in this field), QIAsymphony (a robotics method), and a simple boiling method. Dental plaque was initially collected from three volunteers in the pilot study and then expanded to 12 volunteers in the follow-up study. Bacterial flora was estimated by sequencing the V4 region of 16S rRNA following species-level profiling. Our results indicate that the efficiency of PowerSoil and QIAsymphony was comparable to the boiling method. Therefore, the boiling method may be a promising alternative because of its simplicity, cost effectiveness, and short handling time. Moreover, this method was reliable for estimating bacterial species and could be used in the future to examine the correlation between oral flora and health status. Despite this, differences in the efficiency of DNA extraction for various bacterial species were observed among the three methods. Based on these findings, there is no "gold standard" for DNA extraction. In future, we suggest that the DNA extraction method should be selected on a case-by-case basis considering the aims and specimens of the study.

  10. Evaluation of different pulverisation methods for RNA extraction in squash fruit: lyophilisation, cryogenic mill and mortar grinding.

    PubMed

    Román, Belén; González-Verdejo, Clara I; Peña, Francisco; Nadal, Salvador; Gómez, Pedro

    2012-01-01

    Quality and integrity of RNA are critical for transcription studies in plant molecular biology. In squash fruit and other high water content crops, the grinding of tissue with mortar and pestle in liquid nitrogen fails to produce a homogeneous and fine powered sample desirable to ensure a good penetration of the extraction reagent. To develop an improved pulverisation method to facilitate the homogenisation process of squash fruit tissue prior to RNA extraction without reducing quality and yield of the extracted RNA. Three methods of pulverisation, each followed by the same extraction protocol, were compared. The first approach consisted of the lyophilisation of the sample in order to remove the excess of water before grinding, the second one used a cryogenic mill and the control one a mortar grinding of frozen tissue. The quality of the isolated RNA was tested by carrying out a quantitative real time downstream amplification. In the three situations considered, mean values for A(260) /A(280) indicated minimal interference by proteins and RNA quality indicator (RQI) values were considered appropriate for quantitative real-time polymerase chain reaction (qRT-PCR) amplification. Successful qRT-PCR amplifications were obtained with cDNA isolated with the three protocols. Both apparatus can improve and facilitate the grinding step in the RNA extraction process in zucchini, resulting in isolated RNA of high quality and integrity as revealed by qRT-PCR downstream application. This is apparently the first time that a cryogenic mill has been used to prepare fruit samples for RNA extraction, thereby improving the sampling strategy because the fine powder obtained represents a homogeneous mix of the organ tissue. Copyright © 2012 John Wiley & Sons, Ltd.

  11. A Melting Method for RNA Extraction from the Mucosal Membrane of the Mouse Middle Ear

    PubMed Central

    Seo, Young Joon; Kim, Sung Huhn; Moon, In Seok

    2015-01-01

    Purpose There is much confusion surrounding the methods of RNA extraction from the middle ear mucosa of mice. In this study, we worked to develop a "melting method," which is faster, purer, and more reliable than other methods in common use. Materials and Methods Thirty-two ears were used for this study. Light microscopy with hematoxylin-eosin staining of the bullae, scanning electron microscopy (SEM), spectrophotometer analysis, and reverse transcription polymerase chain reaction were performed before and after melting the half lateral bullae, which were detached from the temporal bone by using a lateral retroauricular approach. Results Each resected half bulla contained a well distributed mucosal membrane. After a TRIzol melting duration of 10-30 minutes, only mucosal marker (MUC5AC) was expressed without bony marker (total osteocalcin). The same results were determined from SEM. Conclusion This melting method, compared with stripping and irrigation methods, is effective and offers an easier, more robust approach to extracting RNA from the middle ear mucosal membranes of mice. PMID:25684001

  12. A protocol for extraction of high-quality RNA and DNA from peanut plant tissues.

    PubMed

    Yin, Dongmei; Liu, Haiying; Zhang, Xingguo; Cui, Dangqun

    2011-10-01

    Peanuts are an increasingly important global food source. However, until recently the lack of effective protocols for the extraction of nucleic acids has made molecular studies of peanut development and maturation difficult. Here, we describe a method to isolate high-quality RNA and DNA from peanut tissue and have successfully applied this method to peanut plant roots, stems, leaves, flowers, and seeds. Spectrophotometric analysis showed that the average yields of total RNA from 100 mg of peanut materials ranged from 24.52 to 74.6 μg, and those of genomic DNA from the same tissues ranged from 23.47 to 57.68 μg. Using this protocol, we obtained OD260/280 values between 1.9 and 2.0 and isolated RNA which could be reverse transcribed in a manner suitable for RT-qPCR and expression analysis. In addition, genomic DNA isolated using this method produced reliable restriction enzyme digestion patterns and could be used for Southern blot hybridization.

  13. Technical note: Comparative analyses of the quality and yield of genomic DNA from invasive and noninvasive, automated and manual extraction methods.

    PubMed

    Foley, C; O'Farrelly, C; Meade, K G

    2011-06-01

    Several new automated methods have recently become available for high-throughput DNA extraction, including the Maxwell 16 System (Promega UK, Southampton, UK). The purpose of this report is to compare automated with manual DNA extraction methods, and invasive with noninvasive sample collection methods, in terms of DNA yield and quality. Milk, blood, and nasal swab samples were taken from 10 cows for DNA extraction. Nasal swabs were also taken from 10 calves and semen samples from 15 bulls for comparative purposes. The Performagene Livestock (DNA Genotek, Kanata, Ontario, Canada) method was compared with similar samples taken from the same animal using manual extraction methods. All samples were analyzed using both the Qubit Quantification Platform (Invitrogen Ltd., Paisley, UK) and NanoDrop spectrophotometer (NanoDrop Technologies, Inc., Wilmington, DE) to accurately assess DNA quality and quantity. In general, the automated Maxwell 16 System performed best, consistently yielding high quantity and quality DNA across the sample range tested. Average yields of 28.7, 10.3, and 19.2 μg of DNA were obtained from 450 μL of blood, 400 μL of milk, and a single straw of semen, respectively. The quality of DNA obtained from buffy coat and from semen was significantly higher with the automated method than with the manual methods (260/280 ratio of 1.9 and 1.8, respectively). Centrifugation of whole blood facilitated the concentration of leukocytes in the buffy coat, which significantly increased DNA yield after manual extraction. The Performagene method also yielded 18.4 and 49.8 μg of high quality (260/280 ratio of 1.8) DNA from the cow and calf nasal samples, respectively. These results show the advantages of noninvasive sample collection and automated methods for high-throughput extraction and biobanking of high quality DNA. Copyright © 2011 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

  14. Automated extraction of information on chemical-P-glycoprotein interactions from the literature.

    PubMed

    Yoshida, Shuya; Yamashita, Fumiyoshi; Ose, Atsushi; Maeda, Kazuya; Sugiyama, Yuichi; Hashida, Mitsuru

    2013-10-28

    Knowledge of the interactions between drugs and transporters is important for drug discovery and development as well as for the evaluation of their clinical safety. We recently developed a text-mining system for the automatic extraction of information on chemical-CYP3A4 interactions from the literature. This system is based on natural language processing and can extract chemical names and their interaction patterns according to sentence context. The present study aimed to extend this system to the extraction of information regarding chemical-transporter interactions. For this purpose, the key verb list designed for cytochrome P450 enzymes was replaced with that for known drug transporters. The performance of the system was then tested by examining the accuracy of information on chemical-P-glycoprotein (P-gp) interactions extracted from randomly selected PubMed abstracts. The system achieved 89.8% recall and 84.2% precision for the identification of chemical names and 71.7% recall and 78.6% precision for the extraction of chemical-P-gp interactions.

  15. [Corrected Title: Solid-Phase Extraction of Polar Compounds from Water] Automated Electrostatics Environmental Chamber

    NASA Technical Reports Server (NTRS)

    Sauer, Richard; Rutz, Jeffrey; Schultz, John

    2005-01-01

    A solid-phase extraction (SPE) process has been developed for removing alcohols, carboxylic acids, aldehydes, ketones, amines, and other polar organic compounds from water. This process can be either a subprocess of a water-reclamation process or a means of extracting organic compounds from water samples for gas-chromatographic analysis. This SPE process is an attractive alternative to an Environmental Protection Administration liquid-liquid extraction process that generates some pollution and does not work in a microgravitational environment. In this SPE process, one forces a water sample through a resin bed by use of positive pressure on the upstream side and/or suction on the downstream side, thereby causing organic compounds from the water to be adsorbed onto the resin. If gas-chromatographic analysis is to be done, the resin is dried by use of a suitable gas, then the adsorbed compounds are extracted from the resin by use of a solvent. Unlike the liquid-liquid process, the SPE process works in both microgravity and Earth gravity. In comparison with the liquid-liquid process, the SPE process is more efficient, extracts a wider range of organic compounds, generates less pollution, and costs less.

  16. RNA.

    ERIC Educational Resources Information Center

    Darnell, James E., Jr.

    1985-01-01

    Ribonucleic acid (RNA) converts genetic information into protein and usually must be processed to serve its function. RNA types, chemical structure, protein synthesis, translation, manufacture, and processing are discussed. Concludes that the first genes might have been spliced RNA and that humans might be closer than bacteria to primitive…

  17. RNA.

    ERIC Educational Resources Information Center

    Darnell, James E., Jr.

    1985-01-01

    Ribonucleic acid (RNA) converts genetic information into protein and usually must be processed to serve its function. RNA types, chemical structure, protein synthesis, translation, manufacture, and processing are discussed. Concludes that the first genes might have been spliced RNA and that humans might be closer than bacteria to primitive…

  18. Comparative Evaluation of a Commercially Available Automated System for Extraction of Viral DNA from Whole Blood: Application to Monitoring of Epstein-Barr Virus and Cytomegalovirus Load ▿

    PubMed Central

    Pillet, Sylvie; Bourlet, Thomas; Pozzetto, Bruno

    2009-01-01

    The NucliSENS easyMAG automated system was compared to the column-based Qiagen method for Epstein-Barr virus (EBV) or cytomegalovirus (CMV) DNA extraction from whole blood before viral load determination using the corresponding R-gene amplification kits. Both extraction techniques exhibited a total agreement of 81.3% for EBV and 87.2% for CMV. PMID:19710270

  19. Yeast tRNA3Leu gene transcribed and spliced in a HeLa cell extract.

    PubMed Central

    Standring, D N; Venegas, A; Rutter, W J

    1981-01-01

    A cloned yeast tRNA3Leu gene containing a 33-base intervening sequence (IVS) is selectively transcribed by a soluble extract from HeLa cells. The 130-nucleotide tRNA3Leu precursor RNA formed is colinear with the gene and contains approximately 4 leader nucleotides and up to 9 trailer nucleotides. The IVS is accurately and efficiently removed by an endogenous HeLa excision-ligase activity to yield the spliced tRNA, the free IVS, and the half-tRNA intermediates. The splicing reaction occurs without prior 5' and 3' maturation of the precursor but, with this exception, this pattern of synthesis and subsequent maturation of the tRNA3Leu precursor conforms to the scheme for tRNA biosynthesis deduced for the xenopus system. Indeed, the two systems utilize similar or identical tRNA3Leu precursors. Our results stress the extraordinary conservation of tRNA biosynthesis in eukaryotes and demonstrate that a HeLa extract provides a useful system for investigating this process. Images PMID:6796956

  20. Automation of static and dynamic non-dispersive liquid phase microextraction. Part 1: Approaches based on extractant drop-, plug-, film- and microflow-formation.

    PubMed

    Alexovič, Michal; Horstkotte, Burkhard; Solich, Petr; Sabo, Ján

    2016-02-04

    Simplicity, effectiveness, swiftness, and environmental friendliness - these are the typical requirements for the state of the art development of green analytical techniques. Liquid phase microextraction (LPME) stands for a family of elegant sample pretreatment and analyte preconcentration techniques preserving these principles in numerous applications. By using only fractions of solvent and sample compared to classical liquid-liquid extraction, the extraction kinetics, the preconcentration factor, and the cost efficiency can be increased. Moreover, significant improvements can be made by automation, which is still a hot topic in analytical chemistry. This review surveys comprehensively and in two parts the developments of automation of non-dispersive LPME methodologies performed in static and dynamic modes. Their advantages and limitations and the reported analytical performances are discussed and put into perspective with the corresponding manual procedures. The automation strategies, techniques, and their operation advantages as well as their potentials are further described and discussed. In this first part, an introduction to LPME and their static and dynamic operation modes as well as their automation methodologies is given. The LPME techniques are classified according to the different approaches of protection of the extraction solvent using either a tip-like (needle/tube/rod) support (drop-based approaches), a wall support (film-based approaches), or microfluidic devices. In the second part, the LPME techniques based on porous supports for the extraction solvent such as membranes and porous media are overviewed. An outlook on future demands and perspectives in this promising area of analytical chemistry is finally given.

  1. Digestion of chrysanthemum stunt viroid by leaf extracts of Capsicum chinense indicates strong RNA-digesting activity.

    PubMed

    Iraklis, Boubourakas; Kanda, Hiroko; Nabeshima, Tomoyuki; Onda, Mayu; Ota, Nao; Koeda, Sota; Hosokawa, Munetaka

    2016-08-01

    CSVd could not infect Nicotiana benthamiana when the plants were pretreated with crude leaf extract of Capsicum chinense 'Sy-2'. C. chinense leaves were revealed to contain strong RNA-digesting activity. Several studies have identified active antiviral and antiviroid agents in plants. Capsicum plants are known to contain antiviral agents, but the mechanism of their activity has not been determined. We aimed to elucidate the mechanism of Capsicum extract's antiviroid activity. Chrysanthemum stunt viroid (CSVd) was inoculated into Nicotiana benthamiana plants before or after treating the plants with a leaf extract of Capsicum chinense 'Sy-2'. CSVd infection was determined using quantitative reverse transcription-polymerase chain reaction (qRT-PCR) 3 weeks after inoculation. When Capsicum extract was sprayed or painted onto N. benthamiana before inoculation, it was effective in preventing infection by CSVd. To evaluate CSVd digestion activity in leaf extracts, CSVd was mixed with leaf extracts of Mirabilis, Phytolacca, Pelargonium and Capsicum. CSVd-digesting activities were examined by quantifying undigested CSVd using qRT-PCR, and RNA gel blotting permitted visualization of the digested CSVd. Only Capsicum leaf extract digested CSVd, and in the Capsicum treatment, small digested CSVd products were detected by RNA gel blot analysis. When the digesting experiment was performed for various cultivars and species of Capsicum, only cultivars of C. chinense showed strong CSVd-digesting activity. Our observations indicated that Capsicum extract contains strong RNA-digesting activity, leading to the conclusion that this activity is the main mechanism for protection from infection by CSVd through spraying or painting before inoculation. To our knowledge, this is the first report of a strong RNA-digesting activity by a plant extract.

  2. Efficient extraction of small and large RNAs in bacteria for excellent total RNA sequencing and comprehensive transcriptome analysis.

    PubMed

    Heera, Rajandas; Sivachandran, Parimannan; Chinni, Suresh V; Mason, Joanne; Croft, Larry; Ravichandran, Manickam; Yin, Lee Su

    2015-12-08

    Next-generation transcriptome sequencing (RNA-Seq) has become the standard practice for studying gene splicing, mutations and changes in gene expression to obtain valuable, accurate biological conclusions. However, obtaining good sequencing coverage and depth to study these is impeded by the difficulties of obtaining high quality total RNA with minimal genomic DNA contamination. With this in mind, we evaluated the performance of Phenol-free total RNA purification kit (Amresco) in comparison with TRI Reagent (MRC) and RNeasy Mini (Qiagen) for the extraction of total RNA of Pseudomonas aeruginosa which was grown in glucose-supplemented (control) and polyethylene-supplemented (growth-limiting condition) minimal medium. All three extraction methods were coupled with an in-house DNase I treatment before the yield, integrity and size distribution of the purified RNA were assessed. RNA samples extracted with the best extraction kit were then sequenced using the Illumina HiSeq 2000 platform. TRI Reagent gave the lowest yield enriched with small RNAs (sRNAs), while RNeasy gave moderate yield of good quality RNA with trace amounts of sRNAs. The Phenol-free kit, on the other hand, gave the highest yield and the best quality RNA (RIN value of 9.85 ± 0.3) with good amounts of sRNAs. Subsequent bioinformatic analysis of the sequencing data revealed that 5435 coding genes, 452 sRNAs and 7 potential novel intergenic sRNAs were detected, indicating excellent sequencing coverage across RNA size ranges. In addition, detection of low abundance transcripts and consistency of their expression profiles across replicates from the same conditions demonstrated the reproducibility of the RNA extraction technique. Amresco's Phenol-free Total RNA purification kit coupled with DNase I treatment yielded the highest quality RNAs containing good ratios of high and low molecular weight transcripts with minimal genomic DNA. These RNA extracts gave excellent non-biased sequencing coverage useful

  3. Sample preparation for avian and porcine influenza virus cDNA amplification simplified: Boiling vs. conventional RNA extraction.

    PubMed

    Fereidouni, Sasan R; Starick, Elke; Ziller, Mario; Harder, Timm C; Unger, Hermann; Hamilton, Keith; Globig, Anja

    2015-09-01

    RNA extraction and purification is a fundamental step that allows for highly sensitive amplification of specific RNA targets in PCR applications. However, commercial extraction kits that are broadly used because of their robustness and high yield of purified RNA are expensive and labor-intensive. In this study, boiling in distilled water or a commercial lysis buffer of different sample matrices containing avian or porcine influenza viruses was tested as an alternative. Real-time PCR (RTqPCR) for nucleoprotein gene fragment was used as read out. Results were compared with freshly extracted RNA by use of a commercial extraction kit. Different batches of virus containing materials, including diluted virus positive allantoic fluid or cell culture supernatant, and avian faecal, cloacal or oropharyngeal swab samples were used in this study. Simple boiling of samples without any additional purification steps can be used as an alternative RNA preparation method to detect influenza A virus nucleoprotein RNA in oropharyngeal swab samples, allantoic fluid or cell-culture supernatant. The boiling method is not applicable for sample matrices containing faecal material. Copyright © 2015 Elsevier B.V. All rights reserved.

  4. ISRU: Automated Water Extraction Ffrom Mars Surface Soils for Sample Return Missions

    NASA Astrophysics Data System (ADS)

    Willson, D.

    2012-06-01

    An ISRU option for Mars sample return vehicles is to employ a Sojourner/MER sized bucket excavation rover that mines and extracts water from the top 5 cm of surface soils and delivers it to an ISRU on the lander. The option is mass competitive.

  5. An automated algorithm for extracting road edges from terrestrial mobile LiDAR data

    NASA Astrophysics Data System (ADS)

    Kumar, Pankaj; McElhinney, Conor P.; Lewis, Paul; McCarthy, Timothy

    2013-11-01

    Terrestrial mobile laser scanning systems provide rapid and cost effective 3D point cloud data which can be used for extracting features such as the road edge along a route corridor. This information can assist road authorities in carrying out safety risk assessment studies along road networks. The knowledge of the road edge is also a prerequisite for the automatic estimation of most other road features. In this paper, we present an algorithm which has been developed for extracting left and right road edges from terrestrial mobile LiDAR data. The algorithm is based on a novel combination of two modified versions of the parametric active contour or snake model. The parameters involved in the algorithm are selected empirically and are fixed for all the road sections. We have developed a novel way of initialising the snake model based on the navigation information obtained from the mobile mapping vehicle. We tested our algorithm on different types of road sections representing rural, urban and national primary road sections. The successful extraction of road edges from these multiple road section environments validates our algorithm. These findings and knowledge provide valuable insights as well as a prototype road edge extraction tool-set, for both national road authorities and survey companies.

  6. Automated extraction of information from the literature on chemical-CYP3A4 interactions.

    PubMed

    Feng, Chunlai; Yamashita, Fumiyoshi; Hashida, Mitsuru

    2007-01-01

    A text mining system is presented for automatically extracting information from the literature on chemical-CYP3A4 interactions (i.e., substrate, induction, inhibition). The system identifies chemicals and CYP3A4 forms according to a combination of name dictionaries and context features. In addition, it transforms sentences into multiple simple clauses each containing a single event and extracts information on chemical-CYP3A4 interactions using a simple but effective pattern matching method based on the order of three keywords (chemicals, CYP3A4, key verbs). Using this system, 2990 relations including 2700 identified interactions with CYP3A4 for 600 chemicals were extracted from a corpus of 2900 PubMed abstracts. In an evaluation test using 100 randomly selected abstracts, it achieved 87.4% recall and 92.3% precision for identification of the chemical name and 85.2% recall and 92.0% precision for the extraction of chemical-CYP3A4 interactions, respectively. This system will be applicable to interactions of chemicals with any functional proteins, such as enzymes and transporters, simply by changing the list of key verbs.

  7. Rapid DNA, RNA and protein extraction protocols optimized for slow continuously growing yeast cultures.

    PubMed

    Sasidharan, Kalesh; Amariei, Cornelia; Tomita, Masaru; Murray, Douglas B

    2012-08-01

    Conventional extraction protocols for yeast have been developed for relatively rapid-growing low cell density cultures of laboratory strains and often do not have the integrity for frequent sampling of cultures. Therefore, these protocols are usually inefficient for cultures under slow growth conditions or of non-laboratory strains. We have developed a combined mechanical and chemical disruption procedure using vigorous bead-beating that can consistently disrupt yeast cells (> 95%), irrespective of cell cycle and metabolic state. Using this disruption technique coupled with quenching, we have developed DNA, RNA and protein extraction protocols that are optimized for a large number of samples from slow-growing high-density industrial yeast cultures. Additionally, sample volume, the use of expensive reagents/enzymes, handling times and incubations were minimized. We have tested the reproducibility of our methods using triplicate/time-series extractions and compared these with commonly used protocols or commercially available kits. Moreover, we utilized a simple flow-cytometric approach to estimate the mitochondrial DNA copy number. Based on the results, our methods have shown higher reproducibility, yield and quality. Copyright © 2012 John Wiley & Sons, Ltd.

  8. Automated Template-based Brain Localization and Extraction for Fetal Brain MRI Reconstruction.

    PubMed

    Tourbier, Sébastien; Velasco-Annis, Clemente; Taimouri, Vahid; Hagmann, Patric; Meuli, Reto; Warfield, Simon K; Cuadra, Meritxell Bach; Gholipour, Ali

    2017-04-10

    Most fetal brain MRI reconstruction algorithms rely only on brain tissue-relevant voxels of low-resolution (LR) images to enhance the quality of inter-slice motion correction and image reconstruction. Consequently the fetal brain needs to be localized and extracted as a first step, which is usually a laborious and time consuming manual or semi-automatic task. We have proposed in this work to use age-matched template images as prior knowledge to automatize brain localization and extraction. This has been achieved through a novel automatic brain localization and extraction method based on robust template-to-slice block matching and deformable slice-to-template registration. Our template-based approach has also enabled the reconstruction of fetal brain images in standard radiological anatomical planes in a common coordinate space. We have integrated this approach into our new reconstruction pipeline that involves intensity normalization, inter-slice motion correction, and super-resolution (SR) reconstruction. To this end we have adopted a novel approach based on projection of every slice of the LR brain masks into the template space using a fusion strategy. This has enabled the refinement of brain masks in the LR images at each motion correction iteration. The overall brain localization and extraction algorithm has shown to produce brain masks that are very close to manually drawn brain masks, showing an average Dice overlap measure of 94.5%. We have also demonstrated that adopting a slice-to-template registration and propagation of the brain mask slice-by-slice leads to a significant improvement in brain extraction performance compared to global rigid brain extraction and consequently in the quality of the final reconstructed images. Ratings performed by two expert observers show that the proposed pipeline can achieve similar reconstruction quality to reference reconstruction based on manual slice-by-slice brain extraction. The proposed brain mask refinement and

  9. Myristica fragrans Houtt. methanolic extract induces apoptosis in a human leukemia cell line through SIRT1 mRNA downregulation.

    PubMed

    Chirathaworn, Chintana; Kongcharoensuntorn, Wisatre; Dechdoungchan, Thitiporn; Lowanitchapat, Alisa; Sa-nguanmoo, Pattaratida; Poovorawan, Yong

    2007-11-01

    Myristica fragrans Houtt. (nutmeg) contains antibacterial, antiviral and anti-cancer activities. However the mechanisms underlying those activities have not been clearly explained. To study the effect of Myristica fragrans Houtt. methanolic extract on Jurkat human leukemia T cell line. Methanol extract of Myristica fragrans Houtt. (Myristicaceae) was used to study the effect on Jurkat cell metabolic activity using an MTT assay and on apoptosis using annexin V staining. Expression of SIRT1 gene was determined by RT-PCR. At the concentrations 50 and 100 ig/mL, the methanol extract of Myristica fragrans Houtt significantly inhibited Jurkat cell proliferation and induced apoptosis as detected by annexin V staining. Downregulation of SIRT1 mRNA expression in Jurkat cells was observed even when the amount of methanol extract was 10 microg/mL. Methanol extract of Myristica fragrans Houtt induced apoptosis of Jurkat leukemia T cell line in a mechanisms involving SIRTI mRNA downregulation.

  10. Using mobile laser scanning data for automated extraction of road markings

    NASA Astrophysics Data System (ADS)

    Guan, Haiyan; Li, Jonathan; Yu, Yongtao; Wang, Cheng; Chapman, Michael; Yang, Bisheng

    2014-01-01

    A mobile laser scanning (MLS) system allows direct collection of accurate 3D point information in unprecedented detail at highway speeds and at less than traditional survey costs, which serves the fast growing demands of transportation-related road surveying including road surface geometry and road environment. As one type of road feature in traffic management systems, road markings on paved roadways have important functions in providing guidance and information to drivers and pedestrians. This paper presents a stepwise procedure to recognize road markings from MLS point clouds. To improve computational efficiency, we first propose a curb-based method for road surface extraction. This method first partitions the raw MLS data into a set of profiles according to vehicle trajectory data, and then extracts small height jumps caused by curbs in the profiles via slope and elevation-difference thresholds. Next, points belonging to the extracted road surface are interpolated into a geo-referenced intensity image using an extended inverse-distance-weighted (IDW) approach. Finally, we dynamically segment the geo-referenced intensity image into road-marking candidates with multiple thresholds that correspond to different ranges determined by point-density appropriate normality. A morphological closing operation with a linear structuring element is finally used to refine the road-marking candidates by removing noise and improving completeness. This road-marking extraction algorithm is comprehensively discussed in the analysis of parameter sensitivity and overall performance. An experimental study performed on a set of road markings with ground-truth shows that the proposed algorithm provides a promising solution to the road-marking extraction from MLS data.

  11. Quantitative analysis of ex vivo colorectal epithelium using an automated feature extraction algorithm for microendoscopy image data

    PubMed Central

    Prieto, Sandra P.; Lai, Keith K.; Laryea, Jonathan A.; Mizell, Jason S.; Muldoon, Timothy J.

    2016-01-01

    Abstract. Qualitative screening for colorectal polyps via fiber bundle microendoscopy imaging has shown promising results, with studies reporting high rates of sensitivity and specificity, as well as low interobserver variability with trained clinicians. A quantitative image quality control and image feature extraction algorithm (QFEA) was designed to lessen the burden of training and provide objective data for improved clinical efficacy of this method. After a quantitative image quality control step, QFEA extracts field-of-view area, crypt area, crypt circularity, and crypt number per image. To develop and validate this QFEA, a training set of microendoscopy images was collected from freshly resected porcine colon epithelium. The algorithm was then further validated on ex vivo image data collected from eight human subjects, selected from clinically normal appearing regions distant from grossly visible tumor in surgically resected colorectal tissue. QFEA has proven flexible in application to both mosaics and individual images, and its automated crypt detection sensitivity ranges from 71 to 94% despite intensity and contrast variation within the field of view. It also demonstrates the ability to detect and quantify differences in grossly normal regions among different subjects, suggesting the potential efficacy of this approach in detecting occult regions of dysplasia. PMID:27335893

  12. Fully automated Liquid Extraction-Based Surface Sampling and Ionization Using a Chip-Based Robotic Nanoelectrospray Platform

    SciTech Connect

    Kertesz, Vilmos; Van Berkel, Gary J

    2010-01-01

    A fully automated liquid extraction-based surface sampling device utilizing an Advion NanoMate chip-based infusion nanoelectrospray ionization system is reported. Analyses were enabled for discrete spot sampling by using the Advanced User Interface of the current commercial control software. This software interface provided the parameter control necessary for the NanoMate robotic pipettor to both form and withdraw a liquid microjunction for sampling from a surface. The system was tested with three types of analytically important sample surface types, viz., spotted sample arrays on a MALDI plate, dried blood spots on paper, and whole-body thin tissue sections from drug dosed mice. The qualitative and quantitative data were consistent with previous studies employing other liquid extraction-based surface sampling techniques. The successful analyses performed here utilized the hardware and software elements already present in the NanoMate system developed to handle and analyze liquid samples. Implementation of an appropriate sample (surface) holder, a solvent reservoir, faster movement of the robotic arm, finer control over solvent flow rate when dispensing and retrieving the solution at the surface, and the ability to select any location on a surface to sample from would improve the analytical performance and utility of the platform.

  13. A Simple Method for Automated Solid Phase Extraction of Water Samples for Immunological Analysis of Small Pollutants.

    PubMed

    Heub, Sarah; Tscharner, Noe; Kehl, Florian; Dittrich, Petra S; Follonier, Stéphane; Barbe, Laurent

    2016-01-01

    A new method for solid phase extraction (SPE) of environmental water samples is proposed. The developed prototype is cost-efficient and user friendly, and enables to perform rapid, automated and simple SPE. The pre-concentrated solution is compatible with analysis by immunoassay, with a low organic solvent content. A method is described for the extraction and pre-concentration of natural hormone 17β-estradiol in 100 ml water samples. Reverse phase SPE is performed with octadecyl-silica sorbent and elution is done with 200 µl of methanol 50% v/v. Eluent is diluted by adding di-water to lower the amount of methanol. After preparing manually the SPE column, the overall procedure is performed automatically within 1 hr. At the end of the process, estradiol concentration is measured by using a commercial enzyme-linked immune-sorbent assay (ELISA). 100-fold pre-concentration is achieved and the methanol content in only 10% v/v. Full recoveries of the molecule are achieved with 1 ng/L spiked de-ionized and synthetic sea water samples.

  14. Automated reference region extraction and population-based input function for brain [11C]TMSX PET image analyses

    PubMed Central

    Rissanen, Eero; Tuisku, Jouni; Luoto, Pauliina; Arponen, Eveliina; Johansson, Jarkko; Oikonen, Vesa; Parkkola, Riitta; Airas, Laura; Rinne, Juha O

    2015-01-01

    [11C]TMSX ([7-N-methyl-11C]-(E)-8-(3,4,5-trimethoxystyryl)-1,3,7-trimethylxanthine) is a selective adenosine A2A receptor (A2AR) radioligand. In the central nervous system (CNS), A2AR are linked to dopamine D2 receptor function in striatum, but they are also important modulators of inflammation. The golden standard for kinetic modeling of brain [11C]TMSX positron emission tomography (PET) is to obtain arterial input function via arterial blood sampling. However, this method is laborious, prone to errors and unpleasant for study subjects. The aim of this work was to evaluate alternative input function acquisition methods for brain [11C]TMSX PET imaging. First, a noninvasive, automated method for the extraction of gray matter reference region using supervised clustering (SCgm) was developed. Second, a method for obtaining a population-based arterial input function (PBIF) was implemented. These methods were created using data from 28 study subjects (7 healthy controls, 12 multiple sclerosis patients, and 9 patients with Parkinson's disease). The results with PBIF correlated well with original plasma input, and the SCgm yielded similar results compared with cerebellum as a reference region. The clustering method for extracting reference region and the population-based approach for acquiring input for dynamic [11C]TMSX brain PET image analyses appear to be feasible and robust methods, that can be applied in patients with CNS pathology. PMID:25370856

  15. Automated hollow fiber microextraction based on two immiscible organic solvents for the extraction of two hormonal drugs.

    PubMed

    Tajik, Mohammad; Yamini, Yadollah; Esrafili, Ali; Ebrahimpour, Behnam

    2015-03-25

    In this research, a rapid efficient and automated instrument based on hollow fiber liquid-phase microextraction (HF-LPME) followed by high performance liquid chromatography (HPLC) with UV-vis detection was applied for the preconcentration and determination of two hormonal drugs (megestrol acetate and levonorgestrel) in water and urinary samples. n-Dodecane was used as the supported liquid membrane (SLM) and methanol was used as the acceptor phase in the hollow fiber lumen. The effects of different parameters such as fiber length, extraction time, stirring rate, and ionic strength on the extraction efficiency were investigated using modified simplex and central composite design as the screening and optimization methods, respectively. The composition effect of SLM and type of acceptor phase were optimized separately. For adjustment of the SLM composition, trioctylphosphine oxide (TOPO) was chosen. Under optimized condition, the calibration curves were linear (r(2)>0.997) in the range of 0.5-200 μg L(-1). LOD for both of the drugs were 0.25 μg L(-1). The applicability of this technique was examined by analyzing drugs in water and urine samples. The relative recoveries of the drugs were in the range of 86.2-102.3% that show the capability of the method for the determination of the drugs in various matrices. Copyright © 2014 Elsevier B.V. All rights reserved.

  16. Progress in automated extraction and purification of in situ 14C from quartz: Results from the Purdue in situ 14C laboratory

    NASA Astrophysics Data System (ADS)

    Lifton, Nathaniel; Goehring, Brent; Wilson, Jim; Kubley, Thomas; Caffee, Marc

    2015-10-01

    Current extraction methods for in situ 14C from quartz [e.g., Lifton et al., (2001), Pigati et al., (2010), Hippe et al., (2013)] are time-consuming and repetitive, making them an attractive target for automation. We report on the status of in situ 14C extraction and purification systems originally automated at the University of Arizona that have now been reconstructed and upgraded at the Purdue Rare Isotope Measurement Laboratory (PRIME Lab). The Purdue in situ 14C laboratory builds on the flow-through extraction system design of Pigati et al. (2010), automating most of the procedure by retrofitting existing valves with external servo-controlled actuators, regulating the pressure of research purity O2 inside the furnace tube via a PID-based pressure controller in concert with an inlet mass flow controller, and installing an automated liquid N2 distribution system, all driven by LabView® software. A separate system for cryogenic CO2 purification, dilution, and splitting is also fully automated, ensuring a highly repeatable process regardless of the operator. We present results from procedural blanks and an intercomparison material (CRONUS-A), as well as results of experiments to increase the amount of material used in extraction, from the standard 5 g to 10 g or above. Results thus far are quite promising with procedural blanks comparable to previous work and significant improvements in reproducibility for CRONUS-A measurements. The latter analyses also demonstrate the feasibility of quantitative extraction of in situ 14C from sample masses up to 10 g. Our lab is now analyzing unknowns routinely, but lowering overall blank levels is the focus of ongoing research.

  17. Detecting and extracting clusters in atom probe data: a simple, automated method using Voronoi cells.

    PubMed

    Felfer, P; Ceguerra, A V; Ringer, S P; Cairney, J M

    2015-03-01

    The analysis of the formation of clusters in solid solutions is one of the most common uses of atom probe tomography. Here, we present a method where we use the Voronoi tessellation of the solute atoms and its geometric dual, the Delaunay triangulation to test for spatial/chemical randomness of the solid solution as well as extracting the clusters themselves. We show how the parameters necessary for cluster extraction can be determined automatically, i.e. without user interaction, making it an ideal tool for the screening of datasets and the pre-filtering of structures for other spatial analysis techniques. Since the Voronoi volumes are closely related to atomic concentrations, the parameters resulting from this analysis can also be used for other concentration based methods such as iso-surfaces. Copyright © 2014 Elsevier B.V. All rights reserved.

  18. Rhythmic brushstrokes distinguish van Gogh from his contemporaries: findings via automated brushstroke extraction.

    PubMed

    Li, Jia; Yao, Lei; Hendriks, Ella; Wang, James Z

    2012-06-01

    Art historians have long observed the highly characteristic brushstroke styles of Vincent van Gogh and have relied on discerning these styles for authenticating and dating his works. In our work, we compared van Gogh with his contemporaries by statistically analyzing a massive set of automatically extracted brushstrokes. A novel extraction method is developed by exploiting an integration of edge detection and clustering-based segmentation. Evidence substantiates that van Gogh's brushstrokes are strongly rhythmic. That is, regularly shaped brushstrokes are tightly arranged, creating a repetitive and patterned impression. We also found that the traits that distinguish van Gogh's paintings in different time periods of his development are all different from those distinguishing van Gogh from his peers. This study confirms that the combined brushwork features identified as special to van Gogh are consistently held throughout his French periods of production (1886-1890).

  19. Automated object extraction from remote sensor image based on adaptive thresholding technique

    NASA Astrophysics Data System (ADS)

    Zhao, Tongzhou; Ma, Shuaijun; Li, Jin; Ming, Hui; Luo, Xiaobo

    2009-10-01

    Detection and extraction of the dim moving small objects in the infrared image sequences is an interesting research area. A system for detection of the dim moving small targets in the IR image sequences is presented, and a new algorithm having high performance for extracting moving small targets in infrared image sequences containing cloud clutter is proposed in the paper. This method can get the better detection precision than some other methods, and two independent units can realize the calculative process. The novelty of the algorithm is that it uses adaptive thresholding technique of the moving small targets in both the spatial domain and temporal domain. The results of experiment show that the algorithm we presented has high ratio of detection precision.

  20. Automated extraction of oscillation parameters for Kepler observations of solar-type stars

    NASA Astrophysics Data System (ADS)

    Huber, D.; Stello, D.; Bedding, T. R.; Chaplin, W. J.; Arentoft, T.; Quirion, P.-O.; Kjeldsen, H.

    2009-10-01

    The recent launch of the Kepler space telescope brings the opportunity to study oscillations systematically in large numbers of solar-like stars. In the framework of the asteroFLAG project, we have developed an automated pipeline to estimate global oscillation parameters, such as the frequency of maximum power (νmax ) and the large frequency spacing (Δν), for a large number of time series. We present an effective method based on the autocorrelation function to find excess power and use a scaling relation to estimate granulation timescales as initial conditions for background modelling. We derive reliable uncertainties for νmax and Δν through extensive simulations. We have tested the pipeline on about 2000 simulated Kepler stars with magnitudes of V ˜ 7-12 and were able to correctly determine νmax and Δν for about half of the sample. For about 20%, the returned large frequency spacing is accurate enough to determine stellar radii to a 1% precision. We conclude that the methods presented here are a promising approach to process the large amount of data expected from Kepler.

  1. Automating identification of avian vocalizations using time-frequency information extracted from the Gabor transform.

    PubMed

    Connor, Edward F; Li, Shidong; Li, Steven

    2012-07-01

    Based on the Gabor transform, a metric is developed and applied to automatically identify bird species from a sample of 568 digital recordings of songs/calls from 67 species of birds. The Gabor frequency-amplitude spectrum and the Gabor time-amplitude profile are proposed as a means to characterize the frequency and time patterns of a bird song. An approach based on template matching where unknown song clips are compared to a library of known song clips is used. After adding noise to simulate the background environment and using an adaptive high-pass filter to de-noise the recordings, the successful identification rate exceeded 93% even at signal-to-noise ratios as low as 5 dB. Bird species whose songs/calls were dominated by low frequencies were more difficult to identify than species whose songs were dominated by higher frequencies. The results suggest that automated identification may be practical if comprehensive libraries of recordings that encompass the vocal variation within species can be assembled.

  2. Quantification of lung tumor rotation with automated landmark extraction using orthogonal cine MRI images

    NASA Astrophysics Data System (ADS)

    Paganelli, Chiara; Lee, Danny; Greer, Peter B.; Baroni, Guido; Riboldi, Marco; Keall, Paul

    2015-09-01

    The quantification of tumor motion in sites affected by respiratory motion is of primary importance to improve treatment accuracy. To account for motion, different studies analyzed the translational component only, without focusing on the rotational component, which was quantified in a few studies on the prostate with implanted markers. The aim of our study was to propose a tool able to quantify lung tumor rotation without the use of internal markers, thus providing accurate motion detection close to critical structures such as the heart or liver. Specifically, we propose the use of an automatic feature extraction method in combination with the acquisition of fast orthogonal cine MRI images of nine lung patients. As a preliminary test, we evaluated the performance of the feature extraction method by applying it on regions of interest around (i) the diaphragm and (ii) the tumor and comparing the estimated motion with that obtained by (i) the extraction of the diaphragm profile and (ii) the segmentation of the tumor, respectively. The results confirmed the capability of the proposed method in quantifying tumor motion. Then, a point-based rigid registration was applied to the extracted tumor features between all frames to account for rotation. The median lung rotation values were  -0.6   ±   2.3° and  -1.5   ±   2.7° in the sagittal and coronal planes respectively, confirming the need to account for tumor rotation along with translation to improve radiotherapy treatment.

  3. Exploratory normalized difference water indices for semi-automated extraction of Antarctic lake features

    NASA Astrophysics Data System (ADS)

    Jawak, Shridhar D.; Luis, Alvarinho J.

    2016-05-01

    This work presents various normalized difference water indices (NDWI) to delineate lakes from Schirmacher Oasis, East Antarctica, by using a very high resolution WorldView-2 (WV-2) satellite imagery. Schirmacher oasis region hosts a number of fresh as well as saline water lakes, such as epishelf lakes, ice-free or landlocked lakes, which are completely frozen or semi-frozen and in a ice-free state. Hence, detecting all these types of lakes distinctly on satellite imagery was the major challenge, as the spectral characteristics of various types of lakes were identical to the other land cover targets. Multiband spectral index pixel-based approach is most experimented and recently growing technique because of its unbeatable advantages such as its simplicity and comparatively lesser amount of processing-time. In present study, semiautomatic extraction of lakes in cryospheric region was carried out by designing specific spectral indices. The study utilized number of existing spectral indices to extract lakes but none could deliver satisfactory results and hence we modified NDWI. The potentials of newly added bands in WV-2 satellite imagery was explored by developing spectral indices comprising of Yellow (585 - 625 nm) band, in combination with Blue (450 - 510 nm), Coastal (400 - 450 nm) and Green (510 - 580 nm) bands. For extraction of frozen lakes, use of Yellow (585 - 625 nm) and near-infrared 2 (NIR2) band pair, and Yellow and Green band pair worked well, whereas for ice-free lakes extraction, a combination of Blue and Coastal band yielded appreciable results, when compared with manually digitized data. The results suggest that the modified NDWI approach rendered bias error varying from 1 to 34 m2.

  4. Application of a novel and automated branched DNA in situ hybridization method for the rapid and sensitive localization of mRNA molecules in plant tissues1

    PubMed Central

    Bowling, Andrew J.; Pence, Heather E.; Church, Jeffrey B.

    2014-01-01

    • Premise of the study: A novel branched DNA detection technology, RNAscope in situ hybridization (ISH), originally developed for use on human clinical and animal tissues, was adapted for use in plant tissue in an attempt to overcome some of the limitations associated with traditional ISH assays. • Methods and Results: Zea mays leaf tissue was formaldehyde fixed and paraffin embedded (FFPE) and then probed with the RNAscope ISH assay for two endogenous genes, phosphoenolpyruvate carboxylase (PEPC) and phosphoenolpyruvate carboxykinase (PEPCK). Results from both manual and automated methods showed tissue- and cell-specific mRNA localization patterns expected from these well-studied genes. • Conclusions: RNAscope ISH is a sensitive method that generates high-quality, easily interpretable results from FFPE plant tissues. Automation of the RNAscope method on the Ventana Discovery Ultra platform allows significant advantages for repeatability, reduction in variability, and flexibility of workflow processes. PMID:25202621

  5. Automated Extraction of Buildings and Roads in a Graph Partitioning Framework

    NASA Astrophysics Data System (ADS)

    Ok, A. O.

    2013-10-01

    This paper presents an original unsupervised framework to identify regions belonging to buildings and roads from monocular very high resolution (VHR) satellite images. The proposed framework consists of three main stages. In the first stage, we extract information only related to building regions using shadow evidence and probabilistic fuzzy landscapes. Firstly, the shadow areas cast by building objects are detected and the directional spatial relationship between buildings and their shadows is modelled with the knowledge of illumination direction. Thereafter, each shadow region is handled separately and initial building regions are identified by iterative graph-cuts designed in a two-label partitioning. The second stage of the framework automatically classifies the image into four classes: building, shadow, vegetation, and others. In this step, the previously labelled building regions as well as the shadow and vegetation areas are involved in a four-label graph optimization performed in the entire image domain to achieve the unsupervised classification result. The final stage aims to extend this classification to five classes in which the class road is involved. For that purpose, we extract the regions that might belong to road segments and utilize that information in a final graph optimization. This final stage eventually characterizes the regions belonging to buildings and roads. Experiments performed on seven test images selected from GeoEye-1 VHR datasets show that the presented approach has ability to extract the regions belonging to buildings and roads in a single graph theory framework.

  6. Automated extraction of DNA from blood and PCR setup using a Tecan Freedom EVO liquid handler for forensic genetic STR typing of reference samples.

    PubMed

    Stangegaard, Michael; Frøslev, Tobias G; Frank-Hansen, Rune; Hansen, Anders J; Morling, Niels

    2011-04-01

    We have implemented and validated automated protocols for DNA extraction and PCR setup using a Tecan Freedom EVO liquid handler mounted with the Te-MagS magnetic separation device (Tecan, Männedorf, Switzerland). The protocols were validated for accredited forensic genetic work according to ISO 17025 using the Qiagen MagAttract DNA Mini M48 kit (Qiagen GmbH, Hilden, Germany) from fresh whole blood and blood from deceased individuals. The workflow was simplified by returning the DNA extracts to the original tubes minimizing the risk of misplacing samples. The tubes that originally contained the samples were washed with MilliQ water before the return of the DNA extracts. The PCR was setup in 96-well microtiter plates. The methods were validated for the kits: AmpFℓSTR Identifiler, SGM Plus and Yfiler (Applied Biosystems, Foster City, CA), GenePrint FFFL and PowerPlex Y (Promega, Madison, WI). The automated protocols allowed for extraction and addition of PCR master mix of 96 samples within 3.5h. In conclusion, we demonstrated that (1) DNA extraction with magnetic beads and (2) PCR setup for accredited, forensic genetic short tandem repeat typing can be implemented on a simple automated liquid handler leading to the reduction of manual work, and increased quality and throughput.

  7. Automated sample preparation based on the sequential injection principle. Solid-phase extraction on a molecularly imprinted polymer coupled on-line to high-performance liquid chromatography.

    PubMed

    Theodoridis, Georgios; Zacharis, Constantinos K; Tzanavaras, Paraskevas D; Themelis, Demetrius G; Economou, Anastasios

    2004-03-19

    A molecularly imprinted polymer (MIP) prepared using caffeine, as a template, was validated as a selective sorbent for solid-phase extraction (SPE), within an automated on-line sample preparation method. The polymer produced was packed in a polypropylene cartridge, which was incorporated in a flow system prior to the HPLC analytical instrumentation. The principle of sequential injection was utilised for a rapid automated and efficient SPE procedure on the MIP. Samples, buffers, washing and elution solvents were introduced to the extraction cartridge via a peristaltic pump and a multi-position valve, both controlled by appropriate software developed in-house. The method was optimised in terms of flow rates, extraction time and volume. After extraction, the final eluent from the extraction cartridge was directed to the injection loop and was subsequently analysed on HPLC. The overall set-up facilitated unattended operation, operation and improved both mixing fluidics and method development flexibility. This system may be readily built in the laboratory and can be further used as an automated platform for on-line sample preparation.

  8. Determination of 21 drugs in oral fluid using fully automated supported liquid extraction and UHPLC-MS/MS.

    PubMed

    Valen, Anja; Leere Øiestad, Åse Marit; Strand, Dag Helge; Skari, Ragnhild; Berg, Thomas

    2016-07-28

    Collection of oral fluid (OF) is easy and non-invasive compared to the collection of urine and blood, and interest in OF for drug screening and diagnostic purposes is increasing. A high-throughput ultra-high-performance liquid chromatography-tandem mass spectrometry method for determination of 21 drugs in OF using fully automated 96-well plate supported liquid extraction for sample preparation is presented. The method contains a selection of classic drugs of abuse, including amphetamines, cocaine, cannabis, opioids, and benzodiazepines. The method was fully validated for 200 μL OF/buffer mix using an Intercept OF sampling kit; validation included linearity, sensitivity, precision, accuracy, extraction recovery, matrix effects, stability, and carry-over. Inter-assay precision (RSD) and accuracy (relative error) were <15% and 13 to 5%, respectively, for all compounds at concentrations equal to or higher than the lower limit of quantification. Extraction recoveries were between 58 and 76% (RSD < 8%), except for tetrahydrocannabinol and three 7-amino benzodiazepine metabolites with recoveries between 23 and 33% (RSD between 51 and 52 % and 11 and 25%, respectively). Ion enhancement or ion suppression effects were observed for a few compounds; however, to a large degree they were compensated for by the internal standards used. Deuterium-labelled and (13) C-labelled internal standards were used for 8 and 11 of the compounds, respectively. In a comparison between Intercept and Quantisal OF kits, better recoveries and fewer matrix effects were observed for some compounds using Quantisal. The method is sensitive and robust for its purposes and has been used successfully since February 2015 for analysis of Intercept OF samples from 2600 cases in a 12-month period. Copyright © 2016 John Wiley & Sons, Ltd.

  9. Evaluation of an Automated Information Extraction Tool for Imaging Data Elements to Populate a Breast Cancer Screening Registry.

    PubMed

    Lacson, Ronilda; Harris, Kimberly; Brawarsky, Phyllis; Tosteson, Tor D; Onega, Tracy; Tosteson, Anna N A; Kaye, Abby; Gonzalez, Irina; Birdwell, Robyn; Haas, Jennifer S

    2015-10-01

    Breast cancer screening is central to early breast cancer detection. Identifying and monitoring process measures for screening is a focus of the National Cancer Institute's Population-based Research Optimizing Screening through Personalized Regimens (PROSPR) initiative, which requires participating centers to report structured data across the cancer screening continuum. We evaluate the accuracy of automated information extraction of imaging findings from radiology reports, which are available as unstructured text. We present prevalence estimates of imaging findings for breast imaging received by women who obtained care in a primary care network participating in PROSPR (n = 139,953 radiology reports) and compared automatically extracted data elements to a "gold standard" based on manual review for a validation sample of 941 randomly selected radiology reports, including mammograms, digital breast tomosynthesis, ultrasound, and magnetic resonance imaging (MRI). The prevalence of imaging findings vary by data element and modality (e.g., suspicious calcification noted in 2.6% of screening mammograms, 12.1% of diagnostic mammograms, and 9.4% of tomosynthesis exams). In the validation sample, the accuracy of identifying imaging findings, including suspicious calcifications, masses, and architectural distortion (on mammogram and tomosynthesis); masses, cysts, non-mass enhancement, and enhancing foci (on MRI); and masses and cysts (on ultrasound), range from 0.8 to1.0 for recall, precision, and F-measure. Information extraction tools can be used for accurate documentation of imaging findings as structured data elements from text reports for a variety of breast imaging modalities. These data can be used to populate screening registries to help elucidate more effective breast cancer screening processes.

  10. An energy minimization approach to automated extraction of regular building footprints from airborne LiDAR data

    NASA Astrophysics Data System (ADS)

    He, Y.; Zhang, C.; Fraser, C. S.

    2014-08-01

    This paper presents an automated approach to the extraction of building footprints from airborne LiDAR data based on energy minimization. Automated 3D building reconstruction in complex urban scenes has been a long-standing challenge in photogrammetry and computer vision. Building footprints constitute a fundamental component of a 3D building model and they are useful for a variety of applications. Airborne LiDAR provides large-scale elevation representation of urban scene and as such is an important data source for object reconstruction in spatial information systems. However, LiDAR points on building edges often exhibit a jagged pattern, partially due to either occlusion from neighbouring objects, such as overhanging trees, or to the nature of the data itself, including unavoidable noise and irregular point distributions. The explicit 3D reconstruction may thus result in irregular or incomplete building polygons. In the presented work, a vertex-driven Douglas-Peucker method is developed to generate polygonal hypotheses from points forming initial building outlines. The energy function is adopted to examine and evaluate each hypothesis and the optimal polygon is determined through energy minimization. The energy minimization also plays a key role in bridging gaps, where the building outlines are ambiguous due to insufficient LiDAR points. In formulating the energy function, hard constraints such as parallelism and perpendicularity of building edges are imposed, and local and global adjustments are applied. The developed approach has been extensively tested and evaluated on datasets with varying point cloud density over different terrain types. Results are presented and analysed. The successful reconstruction of building footprints, of varying structural complexity, along with a quantitative assessment employing accurate reference data, demonstrate the practical potential of the proposed approach.

  11. A novel approach on fluid dispensing for a DNA/RNA extraction chip package

    NASA Astrophysics Data System (ADS)

    Xie, Ling; Premachandran, C. S.; Chew, Michelle; Yao, Qiang; Xu, Diao; Pinjala, D.

    2008-02-01

    Micro fluidic package with integrated reservoirs has been developed for DNA /RNA extraction application. A membrane based pump which consists of a reservoir to store reagents and a pin valve to control the fluid is developed to dispense the reagents into the chip. A programmable external actuator is fabricated to dispense the fluid from the membrane pump into the DNA chip. An elastic and high elongation thin rubber membrane is used to seal the membrane pump and at the same time prevent actuator from mixing with different reagents in the micro fluidic package. Break displacement during actuation of membrane pump sealing material is studied with different ratios of PDMS and other types of rubber materials. The fluid flow from the reservoir to the chip is controlled by a pin valve which is activated during the external actuation. A CFD simulation is performed to study the pumping action dusting the external actuation and is validated with experimental results.

  12. A device for automated direct sampling and quantitation from solid-phase sorbent extraction cards by electrospray tandem mass spectrometry.

    PubMed

    Wachs, Timothy; Henion, Jack

    2003-04-01

    A new solid-phase extraction (SPE) device in the 96-well format (SPE Card) has been employed for automated off-line sample preparation of low-volume urine samples. On-line automated analyte elution via SPE and direct quantitation by micro ion spray mass spectrometry is reported. This sample preparation device has the format of a microtiter plate and is molded in a plastic frame which houses 96 separate sandwiched 3M Empore sorbents (0.5-mm-thickness, 8-microm particles) covered on both sides by a microfiber support material. Ninety-six discrete SPE zones, each 7 mm in diameter, are imbedded into the sheet in the conventional 9-mm pitch (spacing) of a 96-well microtiter plate. In this study one-quarter of an SPE Card (24 individual zones) was used merely as a convenience. After automated off-line interference elution of applied human urine from 24 samples, a section of SPE Card is mounted vertically on a computer-controlled X, Y, Z positioner in front of a micro ion spray direct sampling tube equipped with a beveled tip. The beveled tip of this needle robotically penetrates each SPE elution zone (sorbent disk) or stationary phase in a serial fashion. The eluted analytes are sequentially transferred directly to a microelectrosprayer to obtain tandem mass spectrometric (MS/MS) analysis. This strategy precludes any HPLC separation and the associated method development. The quantitative determination of Ritalin (methylphenidate) from fortified human urine samples is demonstrated. A trideuterated internal standard of methylphenidate was used to obtain ion current response ratios between the parent drug and the internal standard. Human control urine samples fortified from 6.6 to 3300 ng/mL (normal therapeutic levels have been determined in other studies to be between 50 and 100 ng/mL urine) were analyzed and a linear calibration curve was obtained with a correlation coefficient of 0.9999, where the precision of the quality control (QC) samples ranged from 9.6% at the 24

  13. Simultaneous DNA-RNA Extraction from Coastal Sediments and Quantification of 16S rRNA Genes and Transcripts by Real-time PCR

    PubMed Central

    Tatti, Enrico; McKew, Boyd A.; Whitby, Corrine; Smith, Cindy J.

    2016-01-01

    Real Time Polymerase Chain Reaction also known as quantitative PCR (q-PCR) is a widely used tool in microbial ecology to quantify gene abundances of taxonomic and functional groups in environmental samples. Used in combination with a reverse transcriptase reaction (RT-q-PCR), it can also be employed to quantify gene transcripts. q-PCR makes use of highly sensitive fluorescent detection chemistries that allow quantification of PCR amplicons during the exponential phase of the reaction. Therefore, the biases associated with 'end-point' PCR detected in the plateau phase of the PCR reaction are avoided. A protocol to quantify bacterial 16S rRNA genes and transcripts from coastal sediments via real-time PCR is provided. First, a method for the co-extraction of DNA and RNA from coastal sediments, including the additional steps required for the preparation of DNA-free RNA, is outlined. Second, a step-by-step guide for the quantification of 16S rRNA genes and transcripts from the extracted nucleic acids via q-PCR and RT-q-PCR is outlined. This includes details for the construction of DNA and RNA standard curves. Key considerations for the use of RT-q-PCR assays in microbial ecology are included. PMID:27341629

  14. Simultaneous DNA-RNA Extraction from Coastal Sediments and Quantification of 16S rRNA Genes and Transcripts by Real-time PCR.

    PubMed

    Tatti, Enrico; McKew, Boyd A; Whitby, Corrine; Smith, Cindy J

    2016-06-11

    Real Time Polymerase Chain Reaction also known as quantitative PCR (q-PCR) is a widely used tool in microbial ecology to quantify gene abundances of taxonomic and functional groups in environmental samples. Used in combination with a reverse transcriptase reaction (RT-q-PCR), it can also be employed to quantify gene transcripts. q-PCR makes use of highly sensitive fluorescent detection chemistries that allow quantification of PCR amplicons during the exponential phase of the reaction. Therefore, the biases associated with 'end-point' PCR detected in the plateau phase of the PCR reaction are avoided. A protocol to quantify bacterial 16S rRNA genes and transcripts from coastal sediments via real-time PCR is provided. First, a method for the co-extraction of DNA and RNA from coastal sediments, including the additional steps required for the preparation of DNA-free RNA, is outlined. Second, a step-by-step guide for the quantification of 16S rRNA genes and transcripts from the extracted nucleic acids via q-PCR and RT-q-PCR is outlined. This includes details for the construction of DNA and RNA standard curves. Key considerations for the use of RT-q-PCR assays in microbial ecology are included.

  15. DNA and RNA Extraction and Quantitative Real-Time PCR-Based Assays for Biogas Biocenoses in an Interlaboratory Comparison

    PubMed Central

    Lebuhn, Michael; Derenkó, Jaqueline; Rademacher, Antje; Helbig, Susanne; Munk, Bernhard; Pechtl, Alexander; Stolze, Yvonne; Prowe, Steffen; Schwarz, Wolfgang H.; Schlüter, Andreas; Liebl, Wolfgang; Klocke, Michael

    2016-01-01

    Five institutional partners participated in an interlaboratory comparison of nucleic acid extraction, RNA preservation and quantitative Real-Time PCR (qPCR) based assays for biogas biocenoses derived from different grass silage digesting laboratory and pilot scale fermenters. A kit format DNA extraction system based on physical and chemical lysis with excellent extraction efficiency yielded highly reproducible results among the partners and clearly outperformed a traditional CTAB/chloroform/isoamylalcohol based method. Analytical purpose, sample texture, consistency and upstream pretreatment steps determine the modifications that should be applied to achieve maximum efficiency in the trade-off between extract purity and nucleic acid recovery rate. RNA extraction was much more variable, and the destination of the extract determines the method to be used. RNA stabilization with quaternary ammonium salts was an as satisfactory approach as flash freezing in liquid N2. Due to co-eluted impurities, spectrophotometry proved to be of limited value for nucleic acid qualification and quantification in extracts obtained with the kit, and picoGreen® based quantification was more trustworthy. Absorbance at 230 nm can be extremely high in the presence of certain chaotropic guanidine salts, but guanidinium isothiocyanate does not affect (q)PCR. Absolute quantification by qPCR requires application of a reliable internal standard for which correct PCR efficiency and Y-intercept values are important and must be reported. PMID:28952569

  16. CAMUR: Knowledge extraction from RNA-seq cancer data through equivalent classification rules

    PubMed Central

    Cestarelli, Valerio; Fiscon, Giulia; Felici, Giovanni; Bertolazzi, Paola; Weitschek, Emanuel

    2016-01-01

    Motivation: Nowadays, knowledge extraction methods from Next Generation Sequencing data are highly requested. In this work, we focus on RNA-seq gene expression analysis and specifically on case–control studies with rule-based supervised classification algorithms that build a model able to discriminate cases from controls. State of the art algorithms compute a single classification model that contains few features (genes). On the contrary, our goal is to elicit a higher amount of knowledge by computing many classification models, and therefore to identify most of the genes related to the predicted class. Results: We propose CAMUR, a new method that extracts multiple and equivalent classification models. CAMUR iteratively computes a rule-based classification model, calculates the power set of the genes present in the rules, iteratively eliminates those combinations from the data set, and performs again the classification procedure until a stopping criterion is verified. CAMUR includes an ad-hoc knowledge repository (database) and a querying tool. We analyze three different types of RNA-seq data sets (Breast, Head and Neck, and Stomach Cancer) from The Cancer Genome Atlas (TCGA) and we validate CAMUR and its models also on non-TCGA data. Our experimental results show the efficacy of CAMUR: we obtain several reliable equivalent classification models, from which the most frequent genes, their relationships, and the relation with a particular cancer are deduced. Availability and implementation: dmb.iasi.cnr.it/camur.php Contact: emanuel@iasi.cnr.it Supplementary information: Supplementary data are available at Bioinformatics online. PMID:26519501

  17. Automated Identification of Medically Important Bacteria by 16S rRNA Gene Sequencing Using a Novel Comprehensive Database, 16SpathDB▿

    PubMed Central

    Woo, Patrick C. Y.; Teng, Jade L. L.; Yeung, Juilian M. Y.; Tse, Herman; Lau, Susanna K. P.; Yuen, Kwok-Yung

    2011-01-01

    Despite the increasing use of 16S rRNA gene sequencing, interpretation of 16S rRNA gene sequence results is one of the most difficult problems faced by clinical microbiologists and technicians. To overcome the problems we encountered in the existing databases during 16S rRNA gene sequence interpretation, we built a comprehensive database, 16SpathDB (http://147.8.74.24/16SpathDB) based on the 16S rRNA gene sequences of all medically important bacteria listed in the Manual of Clinical Microbiology and evaluated its use for automated identification of these bacteria. Among 91 nonduplicated bacterial isolates collected in our clinical microbiology laboratory, 71 (78%) were reported by 16SpathDB as a single bacterial species having >98.0% nucleotide identity with the query sequence, 19 (20.9%) were reported as more than one bacterial species having >98.0% nucleotide identity with the query sequence, and 1 (1.1%) was reported as no match. For the 71 bacterial isolates reported as a single bacterial species, all results were identical to their true identities as determined by a polyphasic approach. For the 19 bacterial isolates reported as more than one bacterial species, all results contained their true identities as determined by a polyphasic approach and all of them had their true identities as the “best match in 16SpathDB.” For the isolate (Gordonibacter pamelaeae) reported as no match, the bacterium has never been reported to be associated with human disease and was not included in the Manual of Clinical Microbiology. 16SpathDB is an automated, user-friendly, efficient, accurate, and regularly updated database for 16S rRNA gene sequence interpretation in clinical microbiology laboratories. PMID:21389154

  18. Automated Extraction of VTE Events From Narrative Radiology Reports in Electronic Health Records: A Validation Study.

    PubMed

    Tian, Zhe; Sun, Simon; Eguale, Tewodros; Rochefort, Christian M

    2017-10-01

    Surveillance of venous thromboembolisms (VTEs) is necessary for improving patient safety in acute care hospitals, but current detection methods are inaccurate and inefficient. With the growing availability of clinical narratives in an electronic format, automated surveillance using natural language processing (NLP) techniques may represent a better method. We assessed the accuracy of using symbolic NLP for identifying the 2 clinical manifestations of VTE, deep vein thrombosis (DVT) and pulmonary embolism (PE), from narrative radiology reports. A random sample of 4000 narrative reports was selected among imaging studies that could diagnose DVT or PE, and that were performed between 2008 and 2012 in a university health network of 5 adult-care hospitals in Montreal (Canada). The reports were coded by clinical experts to identify positive and negative cases of DVT and PE, which served as the reference standard. Using data from the largest hospital (n=2788), 2 symbolic NLP classifiers were trained; one for DVT, the other for PE. The accuracy of these classifiers was tested on data from the other 4 hospitals (n=1212). On manual review, 663 DVT-positive and 272 PE-positive reports were identified. In the testing dataset, the DVT classifier achieved 94% sensitivity (95% CI, 88%-97%), 96% specificity (95% CI, 94%-97%), and 73% positive predictive value (95% CI, 65%-80%), whereas the PE classifier achieved 94% sensitivity (95% CI, 89%-97%), 96% specificity (95% CI, 95%-97%), and 80% positive predictive value (95% CI, 73%-85%). Symbolic NLP can accurately identify VTEs from narrative radiology reports. This method could facilitate VTE surveillance and the evaluation of preventive measures.

  19. Automated identification and geometrical features extraction of individual trees from Mobile Laser Scanning data in Budapest

    NASA Astrophysics Data System (ADS)

    Koma, Zsófia; Székely, Balázs; Folly-Ritvay, Zoltán; Skobrák, Ferenc; Koenig, Kristina; Höfle, Bernhard

    2016-04-01

    Mobile Laser Scanning (MLS) is an evolving operational measurement technique for urban environment providing large amounts of high resolution information about trees, street features, pole-like objects on the street sides or near to motorways. In this study we investigate a robust segmentation method to extract the individual trees automatically in order to build an object-based tree database system. We focused on the large urban parks in Budapest (Margitsziget and Városliget; KARESZ project) which contained large diversity of different kind of tree species. The MLS data contained high density point cloud data with 1-8 cm mean absolute accuracy 80-100 meter distance from streets. The robust segmentation method contained following steps: The ground points are determined first. As a second step cylinders are fitted in vertical slice 1-1.5 meter relative height above ground, which is used to determine the potential location of each single trees trunk and cylinder-like object. Finally, residual values are calculated as deviation of each point from a vertically expanded fitted cylinder; these residual values are used to separate cylinder-like object from individual trees. After successful parameterization, the model parameters and the corresponding residual values of the fitted object are extracted and imported into the tree database. Additionally, geometric features are calculated for each segmented individual tree like crown base, crown width, crown length, diameter of trunk, volume of the individual trees. In case of incompletely scanned trees, the extraction of geometric features is based on fitted circles. The result of the study is a tree database containing detailed information about urban trees, which can be a valuable dataset for ecologist, city planners, planting and mapping purposes. Furthermore, the established database will be the initial point for classification trees into single species. MLS data used in this project had been measured in the framework of

  20. Linearly Supporting Feature Extraction for Automated Estimation of Stellar Atmospheric Parameters

    NASA Astrophysics Data System (ADS)

    Li, Xiangru; Lu, Yu; Comte, Georges; Luo, Ali; Zhao, Yongheng; Wang, Yongjun

    2015-05-01

    We describe a scheme to extract linearly supporting (LSU) features from stellar spectra to automatically estimate the atmospheric parameters {{T}{\\tt{eff} }}, log g, and [Fe/H]. “Linearly supporting” means that the atmospheric parameters can be accurately estimated from the extracted features through a linear model. The successive steps of the process are as follow: first, decompose the spectrum using a wavelet packet (WP) and represent it by the derived decomposition coefficients; second, detect representative spectral features from the decomposition coefficients using the proposed method Least Absolute Shrinkage and Selection Operator (LARS)bs; third, estimate the atmospheric parameters {{T}{\\tt{eff} }}, log g, and [Fe/H] from the detected features using a linear regression method. One prominent characteristic of this scheme is its ability to evaluate quantitatively the contribution of each detected feature to the atmospheric parameter estimate and also to trace back the physical significance of that feature. This work also shows that the usefulness of a component depends on both the wavelength and frequency. The proposed scheme has been evaluated on both real spectra from the Sloan Digital Sky Survey (SDSS)/SEGUE and synthetic spectra calculated from Kurucz's NEWODF models. On real spectra, we extracted 23 features to estimate {{T}{\\tt{eff} }}, 62 features for log g, and 68 features for [Fe/H]. Test consistencies between our estimates and those provided by the Spectroscopic Parameter Pipeline of SDSS show that the mean absolute errors (MAEs) are 0.0062 dex for log {{T}{\\tt{eff} }} (83 K for {{T}{\\tt{eff} }}), 0.2345 dex for log g, and 0.1564 dex for [Fe/H]. For the synthetic spectra, the MAE test accuracies are 0.0022 dex for log {{T}{\\tt{eff} }} (32 K for {{T}{\\tt{eff} }}), 0.0337 dex for log g, and 0.0268 dex for [Fe/H].

  1. A novel method to identify and isolate proliferative inflammatory atrophy (PIA) clusters and to extract high-quality PIA RNA

    PubMed Central

    Wang, Yibing; Hao, Chao; Fu, Bin; Liu, Weipeng; Zhou, Xiaocheng; Zeng, Tao; Guo, Ju; Wang, Gongxian

    2015-01-01

    Epidemiological and histopathological studies have indicated that proliferative inflammatory atrophy (PIA) of the prostate is closely associated with the onset and development of prostate cancer (PCa). However, accurate isolation of PIA still remains a difficult matter, as well as high-quality RNA extraction from isolated PIA. These issues generated a lack of molecular evidence to support the mechanistic explanation proposed for the progression of PIA to PCa. Therefore, the isolation of PIA and the extraction of high-quality RNA from isolated PIA are of great importance to further demonstrate the correlation between PIA and the development of PCa at a molecular level. In this study, clinical samples from radical prostatectomy were stored in liquid nitrogen, PIA was identified by H&E staining of cryosections, PIA clusters were isolated by manual microdissection, total RNA was extracted from the PIA clusters by Trizol, and RNA quality was determined using the Agilent 2100 Bioanalyzer. Our results showed that PIA might be isolated by manual microdissection of cryosections stored in liquid nitrogen from clinical radical prostatectomy and used for extracting high-quality RNA (RIN > 7.5) by Trizol. Therefore, the present study established a valid method to discover molecular evidence in support of the correlation between PIA and the development of PCa. PMID:26097585

  2. A novel method to identify and isolate proliferative inflammatory atrophy (PIA) clusters and to extract high-quality PIA RNA.

    PubMed

    Wang, Yibing; Hao, Chao; Fu, Bin; Liu, Weipeng; Zhou, Xiaocheng; Zeng, Tao; Guo, Ju; Wang, Gongxian

    2015-01-01

    Epidemiological and histopathological studies have indicated that proliferative inflammatory atrophy (PIA) of the prostate is closely associated with the onset and development of prostate cancer (PCa). However, accurate isolation of PIA still remains a difficult matter, as well as high-quality RNA extraction from isolated PIA. These issues generated a lack of molecular evidence to support the mechanistic explanation proposed for the progression of PIA to PCa. Therefore, the isolation of PIA and the extraction of high-quality RNA from isolated PIA are of great importance to further demonstrate the correlation between PIA and the development of PCa at a molecular level. In this study, clinical samples from radical prostatectomy were stored in liquid nitrogen, PIA was identified by H&E staining of cryosections, PIA clusters were isolated by manual microdissection, total RNA was extracted from the PIA clusters by Trizol, and RNA quality was determined using the Agilent 2100 Bioanalyzer. Our results showed that PIA might be isolated by manual microdissection of cryosections stored in liquid nitrogen from clinical radical prostatectomy and used for extracting high-quality RNA (RIN > 7.5) by Trizol. Therefore, the present study established a valid method to discover molecular evidence in support of the correlation between PIA and the development of PCa.

  3. Pedestrian detection in thermal images: An automated scale based region extraction with curvelet space validation

    NASA Astrophysics Data System (ADS)

    Lakshmi, A.; Faheema, A. G. J.; Deodhare, Dipti

    2016-05-01

    Pedestrian detection is a key problem in night vision processing with a dozen of applications that will positively impact the performance of autonomous systems. Despite significant progress, our study shows that performance of state-of-the-art thermal image pedestrian detectors still has much room for improvement. The purpose of this paper is to overcome the challenge faced by the thermal image pedestrian detectors, which employ intensity based Region Of Interest (ROI) extraction followed by feature based validation. The most striking disadvantage faced by the first module, ROI extraction, is the failed detection of cloth insulted parts. To overcome this setback, this paper employs an algorithm and a principle of region growing pursuit tuned to the scale of the pedestrian. The statistics subtended by the pedestrian drastically vary with the scale and deviation from normality approach facilitates scale detection. Further, the paper offers an adaptive mathematical threshold to resolve the problem of subtracting the background while extracting cloth insulated parts as well. The inherent false positives of the ROI extraction module are limited by the choice of good features in pedestrian validation step. One such feature is curvelet feature, which has found its use extensively in optical images, but has as yet no reported results in thermal images. This has been used to arrive at a pedestrian detector with a reduced false positive rate. This work is the first venture made to scrutinize the utility of curvelet for characterizing pedestrians in thermal images. Attempt has also been made to improve the speed of curvelet transform computation. The classification task is realized through the use of the well known methodology of Support Vector Machines (SVMs). The proposed method is substantiated with qualified evaluation methodologies that permits us to carry out probing and informative comparisons across state-of-the-art features, including deep learning methods, with six

  4. SAR matrices: automated extraction of information-rich SAR tables from large compound data sets.

    PubMed

    Wassermann, Anne Mai; Haebel, Peter; Weskamp, Nils; Bajorath, Jürgen

    2012-07-23

    We introduce the SAR matrix data structure that is designed to elucidate SAR patterns produced by groups of structurally related active compounds, which are extracted from large data sets. SAR matrices are systematically generated and sorted on the basis of SAR information content. Matrix generation is computationally efficient and enables processing of large compound sets. The matrix format is reminiscent of SAR tables, and SAR patterns revealed by different categories of matrices are easily interpretable. The structural organization underlying matrix formation is more flexible than standard R-group decomposition schemes. Hence, the resulting matrices capture SAR information in a comprehensive manner.

  5. Automated feature extraction and spatial organization of seafloor pockmarks, Belfast Bay, Maine, USA

    USGS Publications Warehouse

    Andrews, Brian D.; Brothers, Laura L.; Barnhardt, Walter A.

    2010-01-01

    Seafloor pockmarks occur worldwide and may represent millions of m3 of continental shelf erosion, but few numerical analyses of their morphology and spatial distribution of pockmarks exist. We introduce a quantitative definition of pockmark morphology and, based on this definition, propose a three-step geomorphometric method to identify and extract pockmarks from high-resolution swath bathymetry. We apply this GIS-implemented approach to 25 km2 of bathymetry collected in the Belfast Bay, Maine USA pockmark field. Our model extracted 1767 pockmarks and found a linear pockmark depth-to-diameter ratio for pockmarks field-wide. Mean pockmark depth is 7.6 m and mean diameter is 84.8 m. Pockmark distribution is non-random, and nearly half of the field's pockmarks occur in chains. The most prominent chains are oriented semi-normal to the steepest gradient in Holocene sediment thickness. A descriptive model yields field-wide spatial statistics indicating that pockmarks are distributed in non-random clusters. Results enable quantitative comparison of pockmarks in fields worldwide as well as similar concave features, such as impact craters, dolines, or salt pools.

  6. Automated DICOM metadata and volumetric anatomical information extraction for radiation dosimetry

    NASA Astrophysics Data System (ADS)

    Papamichail, D.; Ploussi, A.; Kordolaimi, S.; Karavasilis, E.; Papadimitroulas, P.; Syrgiamiotis, V.; Efstathopoulos, E.

    2015-09-01

    Patient-specific dosimetry calculations based on simulation techniques have as a prerequisite the modeling of the modality system and the creation of voxelized phantoms. This procedure requires the knowledge of scanning parameters and patients’ information included in a DICOM file as well as image segmentation. However, the extraction of this information is complicated and time-consuming. The objective of this study was to develop a simple graphical user interface (GUI) to (i) automatically extract metadata from every slice image of a DICOM file in a single query and (ii) interactively specify the regions of interest (ROI) without explicit access to the radiology information system. The user-friendly application developed in Matlab environment. The user can select a series of DICOM files and manage their text and graphical data. The metadata are automatically formatted and presented to the user as a Microsoft Excel file. The volumetric maps are formed by interactively specifying the ROIs and by assigning a specific value in every ROI. The result is stored in DICOM format, for data and trend analysis. The developed GUI is easy, fast and and constitutes a very useful tool for individualized dosimetry. One of the future goals is to incorporate a remote access to a PACS server functionality.

  7. Automated boundary extraction of the spinal canal in MRI based on dynamic programming.

    PubMed

    Koh, Jaehan; Chaudhary, Vipin; Dhillon, Gurmeet

    2012-01-01

    The spinal cord is the only communication link between the brain and the body. The abnormalities in it can lead to severe pain and sometimes to paralysis. Due to the growing gap between the number of available radiologists and the number of required radiologists, the need for computer-aided diagnosis and characterization is increasing. To ease this gap, we have developed a computer-aided diagnosis and characterization framework in lumbar spine that includes the spinal cord, vertebrae, and intervertebral discs. In this paper, we propose two spinal cord boundary extraction methods that fit into our framework based on dynamic programming in lumbar spine MRI. Our method incorporates the intensity of the image and the gradient of the image into a dynamic programming scheme and works in a fully-automatic fashion. The boundaries generated by our method is compared against reference boundaries in terms of Fréchet distance which is known to be a metric for shape analysis. The experimental results from 65 clinical data show that our method finds the spinal canal boundary correctly achieving a mean Fréchet distance of 13.5 pixels. For almost all data, the extracted boundary falls within the spinal cord. So, it can be used as a landmark when marking background regions and finding regions of interest.

  8. Automated bare earth extraction technique for complex topography in light detection and ranging surveys

    NASA Astrophysics Data System (ADS)

    Stevenson, Terry H.; Magruder, Lori A.; Neuenschwander, Amy L.; Bradford, Brian

    2013-01-01

    Bare earth extraction is an important component to light detection and ranging (LiDAR) data analysis in terms of terrain classification. The challenge in providing accurate digital surface models is augmented when there is diverse topography within the data set or complex combinations of vegetation and built structures. Few existing algorithms can handle substantial terrain diversity without significant editing or user interaction. This effort presents a newly developed methodology that provides a flexible, adaptable tool capable of integrating multiple LiDAR data attributes for an accurate terrain assessment. The terrain extraction and segmentation (TEXAS) approach uses a third-order spatial derivative for each point in the digital surface model to determine the curvature of the terrain rather than rely solely on the slope. The utilization of the curvature has shown to successfully preserve ground points in areas of steep terrain as they typically exhibit low curvature. Within the framework of TEXAS, the contiguous sets of points with low curvatures are grouped into regions using an edge-based segmentation method. The process does not require any user inputs and is completely data driven. This technique was tested on a variety of existing LiDAR surveys, each with varying levels of topographic complexity.

  9. Automated Neuroanatomical Relation Extraction: A Linguistically Motivated Approach with a PVT Connectivity Graph Case Study

    PubMed Central

    Gökdeniz, Erinç; Özgür, Arzucan; Canbeyli, Reşit

    2016-01-01

    Identifying the relations among different regions of the brain is vital for a better understanding of how the brain functions. While a large number of studies have investigated the neuroanatomical and neurochemical connections among brain structures, their specific findings are found in publications scattered over a large number of years and different types of publications. Text mining techniques have provided the means to extract specific types of information from a large number of publications with the aim of presenting a larger, if not necessarily an exhaustive picture. By using natural language processing techniques, the present paper aims to identify connectivity relations among brain regions in general and relations relevant to the paraventricular nucleus of the thalamus (PVT) in particular. We introduce a linguistically motivated approach based on patterns defined over the constituency and dependency parse trees of sentences. Besides the presence of a relation between a pair of brain regions, the proposed method also identifies the directionality of the relation, which enables the creation and analysis of a directional brain region connectivity graph. The approach is evaluated over the manually annotated data sets of the WhiteText Project. In addition, as a case study, the method is applied to extract and analyze the connectivity graph of PVT, which is an important brain region that is considered to influence many functions ranging from arousal, motivation, and drug-seeking behavior to attention. The results of the PVT connectivity graph show that PVT may be a new target of research in mood assessment. PMID:27708573

  10. Simultaneous analysis of cortisol and cortisone in saliva using XLC-MS/MS for fully automated online solid phase extraction.

    PubMed

    Jones, Rachel L; Owen, Laura J; Adaway, Joanne E; Keevil, Brian G

    2012-01-15

    Salivary cortisol measurements are increasingly being used in the investigation of disorders of the hypothalamic-pituitary-adrenal axis. In the salivary gland, cortisol is metabolised to cortisone by the action of 11β-hydroxysteroid dehydrogenase type 2, and cortisone is partly responsible for the variable interference observed in current salivary cortisol immunoassays. The aim of this study was to validate an assay for the simultaneous analysis of salivary cortisol and cortisone using the Spark Holland Symbiosis™ in eXtraction liquid chromatography-tandem mass spectrometry (XLC-MS/MS) mode for fully automated online solid phase extraction (SPE). Saliva samples were diluted in water with the addition of internal standard (d4-cortisol and d7-cortisone). Online SPE was performed using the Spark Holland Symbiosis™ with HySphere™ C18 SPE cartridges and compounds were eluted onto a Phenomenex® C18 guard column attached to a Phenomenex® Onyx monolithic C18 column for chromatography. Mass spectrometry used the Waters® Xevo™ TQ MS in electrospray positive mode. Cortisol and cortisone eluted with their internal standards at 1.95 and 2.17 min, respectively, with a total run time of four minutes. No evidence of ion-suppression was observed. The assay was linear up to 3393 nmol/L for cortisol and 3676 nmol/L for cortisone, with lower limits of quantitation of 0.75 nmol/L and 0.50 nmol/L, respectively. Intra- and inter-assay imprecision was <8.9% for cortisol and <6.5% for cortisone across three levels of internal quality control, with accuracy and recovery within accepted limits. High specificity was demonstrated following interference studies which assessed 29 structurally-related steroids at supra-physiological concentrations. We have successfully validated an assay for the simultaneous analysis of salivary cortisol and cortisone using XLC-MS/MS and fully automated online SPE. The assay benefits from increased specificity compared to immunoassay and minimal

  11. Optimizing RNA Extraction of Renal Papilla Biopsy Tissue in Kidney Stone Formers: A New Methodology for Genomic Study.

    PubMed

    Taguchi, Kazumi; Usawachintachit, Manint; Hamamoto, Shuzo; Unno, Rei; Tzou, David T; Sherer, Benjamin A; Wang, Yongmei; Okada, Atsushi; Stoller, Marshall L; Yasui, Takahiro; Chi, Thomas

    2017-09-01

    Endoscopic tools have provided versatile examination and treatment for kidney stone procedures. Despite endourologists researching urinary stone disease using endoscopes to collect tissue, this tissue collection method is limited. Endoscopically removed tissues are small in size, restricting the types of genome-based examination possible. We investigated a new method of renal papilla biopsy and RNA extraction to establish a genomic research methodology for kidney stone disease. We conducted a prospective multi-institutional study and collected renal papilla specimens from consecutive percutaneous nephrolithotomy and ureteroscopy (URS) cases performed for removal of upper urinary tract stones. Renal papilla tissue was extracted using ureteroscopic biopsy forceps after stone removal. RNA was extracted using two different extraction kits, and their quantity and quality were examined. Additionally, the impact of biopsy on surgical complications was compared between cases performed with and without biopsy by matched case-control analysis adjusted for age, gender, body mass index, bilaterality, and stone burden. A total of 90 biopsies from 49 patients were performed, and the median duration between specimen collection and RNA extraction was 61 days. Both univariate and multivariate analyses showed BIGopsy(®) forceps usage significantly increased the total yield (p = 0.004) and quality (p = 0.001 for A260/280, p = 0.004 for A260/A230) of extracted RNA. Extraction using the RNeasy Micro Kit(®) also improved A260/A230, whereas reduced RNA integrity number of extracted RNA by univariate and multivariate analyses (p = 0.002 and p < 0.001, respectively). Moreover, matched case-control study demonstrated that endoscopic renal papilla biopsy caused no significant surgical complications, including bleeding, decreased stone clearance and hematocrit, and renal dysfunction. Biopsies during URS imparted an average of 20 minutes of procedure time over nonbiopsy

  12. Magnetic bead technology for viral RNA extraction from serum in blood bank screening.

    PubMed

    Albertoni, Guilherme Ambrozio; Arnoni, Carine Prisco; Araujo, Patricia Regina Barboza; Andrade, Sheila Siqueira; Carvalho, Fabrício Oliveira; Girão, Manoel João Batista Castello; Schor, Nestor; Barreto, José Augusto

    2011-01-01

    Nucleic acid amplification testing (NAT) was recently recommended by Brazilian legislation and has been implemented at some blood banks in the city of São Paulo, Brazil, in an attempt to reduce blood-born transmission of human immunodeficiency virus (HIV) and hepatitis C virus. Manual magnetic particle-based extraction methods for HIV and HCV viral nucleic acids were evaluated in combination with detection by reverse transcriptase - polymerase chain reaction (RT-PCR) one-step. Blood donor samples were collected from January 2010 to September 2010, and minipools of them were submitted to testing. ELISA was used for the analysis of anti-HCV/HIV antibodies. Detection and amplification of viral RNA was performed using real-time PCR. Out of 20.808 samples screened, 53 samples (29 for HCV and 24 for HIV) were confirmed as positive by serological and NAT methods. The manual magnetic bead-based extraction in combination with real-time PCR detection can be used to routinely screen blood donation for viremic donors to further increase the safety of blood products.

  13. A total extract dot blot hybridization procedure for mRNA quantitation in small samples of tissues or cultured cells.

    PubMed

    Grimes, A; McArdle, H J; Mercer, J F

    1988-08-01

    A simple method for the estimation of specific mRNA concentrations in small tissue samples (as little as 1 mg) or cultured cells (lower limit 10(5) cells) is described. Guanidine hydrochloride extracts of whole cells or tissues are applied directly onto nitrocellulose and hybridized with the appropriate nick-translated probe. Loading according to DNA content allows expression of the result as concentration per cell. Hybridizing with a ribosomal RNA probe allows expression of results relative to rRNA and estimation of the RNA/DNA ratio in the sample. We describe the application of this procedure to the measurement of ceruloplasmin mRNA in tissues and cultured hepatocytes.

  14. A novel approach for automated shoreline extraction from remote sensing images using low level programming

    NASA Astrophysics Data System (ADS)

    Rigos, Anastasios; Vaiopoulos, Aristidis; Skianis, George; Tsekouras, George; Drakopoulos, Panos

    2015-04-01

    Tracking coastline changes is a crucial task in the context of coastal management and synoptic remotely sensed data has become an essential tool for this purpose. In this work, and within the framework of BeachTour project, we introduce a new method for shoreline extraction from high resolution satellite images. It was applied on two images taken by the WorldView-2 satellite (7 channels, 2m resolution) during July 2011 and August 2014. The location is the well-known tourist destination of Laganas beach spanning 5 km along the southern part of Zakynthos Island, Greece. The atmospheric correction was performed with the ENVI FLAASH procedure and the final images were validated against hyperspectral field measurements. Using three channels (CH2=blue, CH3=green and CH7=near infrared) the Modified Redness Index image was calculated according to: MRI=(CH7)2/[CH2x(CH3)3]. MRI has the property that its value keeps increasing as the water becomes shallower. This is followed by an abrupt reduction trend at the location of the wet sand up to the point where the dry shore face begins. After that it remains low-valued throughout the beach zone. Images based on this index were used for the shoreline extraction process that included the following steps: a) On the MRI based image, only an area near the shoreline was kept (this process is known as image masking). b) On the masked image the Canny edge detector operator was applied. c) Of all edges discovered on step (b) only the biggest was kept. d) If the line revealed on step (c) was unacceptable, i.e. not defining the shoreline or defining only part of it, then either more than one areas on step (c) were kept or on the MRI image the pixel values were bound in a particular interval [Blow, Bhigh] and only the ones belonging in this interval were kept. Then, steps (a)-(d) were repeated. Using this method, which is still under development, we were able to extract the shoreline position and reveal its changes during the 3-year period

  15. Advancing forensic RNA typing: On non-target secretions, a nasal mucosa marker, a differential co-extraction protocol and the sensitivity of DNA and RNA profiling.

    PubMed

    van den Berge, Margreet; Bhoelai, Bryan; Harteveld, Joyce; Matai, Anuska; Sijen, Titia

    2016-01-01

    The forensic identification of human body fluids and tissues by means of messenger RNA (mRNA) profiling is a long studied methodology that is increasingly applied to casework samples. Previously, we have described an mRNA multiplex system that targets blood, saliva, semen, menstrual secretion, vaginal mucosa and skin (Lindenbergh et al. and van den Berge et al.). In this study we consider various topics to improve this mRNA profiling system or its use and adapt the method accordingly. Bodily secretions that may be encountered at a crime scene whilst not targeted by the multiplex-id est nasal mucosa, sweat, tears, faeces and urine-were examined for false positive signals. The results prompted us to identify a nasal mucosa marker that allows the discrimination of nasal mucosa from saliva or vaginal mucosa and nosebleed blood from peripheral blood. An updated version of the multiplex was prepared to which the nasal mucosa marker was added and in which markers for semen, vaginal mucosa and blood were replaced. Lactobacillus markers were regarded unsuitable as replacement for vaginal mucosa mRNA markers because of background signals on penile swabs that appeared devoid of female DNA. Furthermore, we provide approaches to deal with highly unbalanced mixtures. First, a differential extraction protocol was incorporated into a co-extraction protocol to allow DNA and RNA analysis of separated non-sperm and sperm fractions. In a second approach, besides the standard multiplex, a customized multiplex is used which excludes markers for prevailing cell types. This allows the use of lower cDNA inputs for the prevailing cell types and higher inputs for cell types that appear masked. Additionally, we assessed the relation between the percentage of alleles or markers detected in DNA or RNA profiles when decreasing sample amounts are analysed. While blood, saliva, semen and menstrual secretion show the trend that DNA profiling is more sensitive than RNA profiling, the reverse is seen

  16. An automated method of on-line extraction coupled with flow injection and capillary electrophoresis for phytochemical analysis.

    PubMed

    Chen, Hongli; Ding, Xiuping; Wang, Min; Chen, Xingguo

    2010-11-01

    In this study, an automated system for phytochemical analysis was successfully fabricated for the first time in our laboratory. The system included on-line decocting, filtering, cooling, sample introducing, separation, and detection, which greatly simplified the sample preparation and shortened the analysis time. Samples from the decoction extract were drawn every 5 min through an on-line filter and a condenser pipe to the sample loop from which 20-μL samples were injected into the running buffer and transported into a split-flow interface coupling the flow injection and capillary electrophoresis systems. The separation of glycyrrhetinic acid (GTA) and glycyrrhizic acid (GA) took less than 5 min by using a 10 mM borate buffer (adjusted pH to 8.8) and +10 kV voltage. Calibration curves showed good linearity with correlation coefficients (R) more than 0.9991. The intra-day repeatabilities (n = 5, expressed as relative standard deviation) of the proposed system, obtained using GTA and GA standards, were 1.1% and 0.8% for migration time and 0.7% and 0.9% for peak area, respectively. The mean recoveries of GTA and GA in the off-line extract of Glycyrrhiza uralensis Fisch root were better than 99.0%. The limits of detection (signal-to-noise ratio = 3) of the proposed method were 6.2 μg/mL and 6.9 μg/mL for GTA and GA, respectively. The dynamic changes of GTA and GA on the decoction time were obtained during the on-line decoction process of Glycyrrhiza uralensis Fisch root.

  17. Automated extraction of absorption features from Airborne Visible/Infrared Imaging Spectrometer (AVIRIS) and Geophysical and Environmental Research Imaging Spectrometer (GERIS) data

    NASA Technical Reports Server (NTRS)

    Kruse, Fred A.; Calvin, Wendy M.; Seznec, Olivier

    1988-01-01

    Automated techniques were developed for the extraction and characterization of absorption features from reflectance spectra. The absorption feature extraction algorithms were successfully tested on laboratory, field, and aircraft imaging spectrometer data. A suite of laboratory spectra of the most common minerals was analyzed and absorption band characteristics tabulated. A prototype expert system was designed, implemented, and successfully tested to allow identification of minerals based on the extracted absorption band characteristics. AVIRIS spectra for a site in the northern Grapevine Mountains, Nevada, have been characterized and the minerals sericite (fine grained muscovite) and dolomite were identified. The minerals kaolinite, alunite, and buddingtonite were identified and mapped for a site at Cuprite, Nevada, using the feature extraction algorithms on the new Geophysical and Environmental Research 64 channel imaging spectrometer (GERIS) data. The feature extraction routines (written in FORTRAN and C) were interfaced to the expert system (written in PROLOG) to allow both efficient processing of numerical data and logical spectrum analysis.

  18. Automated and portable solid phase extraction platform for immuno-detection of 17β-estradiol in water.

    PubMed

    Heub, Sarah; Tscharner, Noe; Monnier, Véronique; Kehl, Florian; Dittrich, Petra S; Follonier, Stéphane; Barbe, Laurent

    2015-02-13

    A fully automated and portable system for solid phase extraction (SPE) has been developed for the analysis of the natural hormone 17β-estradiol (E2) in environmental water by enzyme linked immuno-sorbent assay (ELISA). The system has been validated with de-ionized and artificial sea water as model samples and allowed for pre-concentration of E2 at levels of 1, 10 and 100 ng/L with only 100 ml of sample. Recoveries ranged from 24±3% to 107±6% depending on the concentration and sample matrix. The method successfully allowed us to determine the concentration of two seawater samples. A concentration of 15.1±0.3 ng/L of E2 was measured in a sample obtained from a food production process, and 8.8±0.7 ng/L in a sample from the Adriatic Sea. The system would be suitable for continuous monitoring of water quality as it is user friendly, and as the method is reproducible and totally compatible with the analysis of water sample by simple immunoassays and other detection methods such as biosensors.

  19. Automated extraction and quantitation of oncogenic HPV genotypes from cervical samples by a real-time PCR-based system.

    PubMed

    Broccolo, Francesco; Cocuzza, Clementina E

    2008-03-01

    Accurate laboratory assays for the diagnosis of persistent oncogenic HPV infection are being recognized increasingly as essential for clinical management of women with cervical precancerous lesions. HPV viral load has been suggested to be a surrogate marker of persistent infection. Four independent real-time quantitative TaqMan PCR assays were developed for: HPV-16, -31, -18 and/or -45 and -33 and/or -52, -58, -67. The assays had a wide dynamic range of detection and a high degree of accuracy, repeatability and reproducibility. In order to minimize material and hands-on time, automated nucleic acid extraction was performed using a 96-well plate format integrated into a robotic liquid handler workstation. The performance of the TaqMan assays for HPV identification was assessed by comparing results with those obtained by means of PCR using consensus primers (GP5+/GP6+) and sequencing (296 samples) and INNO-LiPA analysis (31 samples). Good agreement was found generally between results obtained by real-time PCR assays and GP(+)-PCR system (kappa statistic=0.91). In conclusion, this study describes four newly developed real-time PCR assays that provide a reliable and high-throughput method for detection of not only HPV DNA but also HPV activity of the most common oncogenic HPV types in cervical specimens.

  20. Dried Blood Spot Proteomics: Surface Extraction of Endogenous Proteins Coupled with Automated Sample Preparation and Mass Spectrometry Analysis

    NASA Astrophysics Data System (ADS)

    Martin, Nicholas J.; Bunch, Josephine; Cooper, Helen J.

    2013-08-01

    Dried blood spots offer many advantages as a sample format including ease and safety of transport and handling. To date, the majority of mass spectrometry analyses of dried blood spots have focused on small molecules or hemoglobin. However, dried blood spots are a potentially rich source of protein biomarkers, an area that has been overlooked. To address this issue, we have applied an untargeted bottom-up proteomics approach to the analysis of dried blood spots. We present an automated and integrated method for extraction of endogenous proteins from the surface of dried blood spots and sample preparation via trypsin digestion by use of the Advion Biosciences Triversa Nanomate robotic platform. Liquid chromatography tandem mass spectrometry of the resulting digests enabled identification of 120 proteins from a single dried blood spot. The proteins identified cross a concentration range of four orders of magnitude. The method is evaluated and the results discussed in terms of the proteins identified and their potential use as biomarkers in screening programs.

  1. Exposing Exposure: Automated Anatomy-specific CT Radiation Exposure Extraction for Quality Assurance and Radiation Monitoring

    PubMed Central

    Warden, Graham I.; Farkas, Cameron E.; Ikuta, Ichiro; Prevedello, Luciano M.; Andriole, Katherine P.; Khorasani, Ramin

    2012-01-01

    Purpose: To develop and validate an informatics toolkit that extracts anatomy-specific computed tomography (CT) radiation exposure metrics (volume CT dose index and dose-length product) from existing digital image archives through optical character recognition of CT dose report screen captures (dose screens) combined with Digital Imaging and Communications in Medicine attributes. Materials and Methods: This institutional review board–approved HIPAA-compliant study was performed in a large urban health care delivery network. Data were drawn from a random sample of CT encounters that occurred between 2000 and 2010; images from these encounters were contained within the enterprise image archive, which encompassed images obtained at an adult academic tertiary referral hospital and its affiliated sites, including a cancer center, a community hospital, and outpatient imaging centers, as well as images imported from other facilities. Software was validated by using 150 randomly selected encounters for each major CT scanner manufacturer, with outcome measures of dose screen retrieval rate (proportion of correctly located dose screens) and anatomic assignment precision (proportion of extracted exposure data with correctly assigned anatomic region, such as head, chest, or abdomen and pelvis). The 95% binomial confidence intervals (CIs) were calculated for discrete proportions, and CIs were derived from the standard error of the mean for continuous variables. After validation, the informatics toolkit was used to populate an exposure repository from a cohort of 54 549 CT encounters; of which 29 948 had available dose screens. Results: Validation yielded a dose screen retrieval rate of 99% (597 of 605 CT encounters; 95% CI: 98%, 100%) and an anatomic assignment precision of 94% (summed DLP fraction correct 563 in 600 CT encounters; 95% CI: 92%, 96%). Patient safety applications of the resulting data repository include benchmarking between institutions, CT protocol quality

  2. Hyphenating Centrifugal Partition Chromatography with Nuclear Magnetic Resonance through Automated Solid Phase Extraction.

    PubMed

    Bisson, Jonathan; Brunel, Marion; Badoc, Alain; Da Costa, Grégory; Richard, Tristan; Mérillon, Jean-Michel; Waffo-Téguo, Pierre

    2016-10-18

    Centrifugal partition chromatography (CPC) and all countercurrent separation apparatus provide chemists with efficient ways to work with complex matrixes, especially in the domain of natural products. However, despite the great advances provided by these techniques, more efficient ways of analyzing the output flow would bring further enhancement. This study describe a hyphenated approach made by coupling NMR with CPC through a hybrid-indirect coupling made possible by using a solid phase extraction (SPE) apparatus intended for high-pressure liquid chromatography (HPLC)-NMR hyphenation. Some hardware changes were needed to adapt the incompatible flow-rates and a reverse-engineering approach that led to the specific software required to control the apparatus. 1D (1)HNMR and (1)H-(1)H correlation spectroscopy (COSY) spectra were acquired in reasonable time without the need for any solvent-suppression method thanks to the SPE nitrogen drying step. The reduced usage of expensive deuterated solvents from several hundreds of milliliters to the milliliter order is the major improvement of this approach compared to the previously published ones.

  3. Automated quantification of distributed landslide movement using circular tree trunks extracted from terrestrial laser scan data

    NASA Astrophysics Data System (ADS)

    Conner, Jeremy C.; Olsen, Michael J.

    2014-06-01

    This manuscript presents a novel algorithm to automatically detect landslide movement in a forested area using displacements of tree trunks distributed across the landslide surveyed repeatedly using terrestrial laser scanning (TLS). Common landslide monitoring techniques include: inclinometers, global position system (GPS), and interferometric synthetic aperture radar (InSAR). While these techniques provide valuable data for monitoring landslides, they can be difficult to apply with adequate spatial or temporal resolution needed to understand complex landslides, specifically in forested environments. Comparison of the center coordinates (determined via least-squares fit of the TLS data) of a cross section of the tree trunk between consecutive surveys enable quantification of landslide movement rates, which can be used to analyze patterns of landslide displacement. The capabilities of this new methodology were tested through a case-study analyzing the Johnson Creek Landslide, a complex, quick moving coastal landslide, which has proven difficult to monitor using other techniques. A parametric analysis of fitting thresholds was also conducted to determine the reliability of tree trunk displacements calculated and the number of features that were extracted. The optimal parameters in selecting trees for movement analysis were found to be less than 1.5 cm for the RMS residuals of the circle fit and less than 1.0 cm for the difference in the calculated tree radii between epochs.

  4. Evaluation of different RNA-extraction kits for sensitive detection of hepatitis A virus in strawberry samples.

    PubMed

    Bianchi, Silvia; Dal Vecchio, Anna; Vilariño, María Luz; Romalde, Jesús L

    2011-02-01

    The efficiency of different commercial RNA extraction kits for the detection of hepatitis A virus (HAV) from seeded strawberry samples was assessed by standard RT-PCR and real time RT-PCR (RT-qPCR). The best results with standard RT-PCR were achieved with Aurum™ Total RNA extraction kit (BioRad), obtaining a detection limit of 5-6.25 pfu/mg of tissue. A slightly lower sensitivity was rendered by the RNeasy® Plant mini kit (Qiagen) (10-12.5 pfu/mg tissue), while the Total Quick RNA Cells and Tissues kit version mini (Talent) rendered a detection limit of 50-100 pfu/mg of tissue. The other tested commercial kits showed worse detection limits (>500 pfu/mg). With RT-qPCR and ten fold diluted RNA all the kits showed an increase of sensitivity, detecting the kits from Qiagen, Talent and BioRad down to 0.05 pfu/mg of strawberry homogenate. These findings indicate that the use of Aurum™ Total RNA extraction kit, with standard RT-PCR technique or RT-qPCR, can not only be labor and time saving but also helpful to improve the sensitivity for the HAV detection from fruits and to facilitate the standardization of detection methods among laboratories.

  5. High-throughput pharmacokinetics screen of VLA-4 antagonists by LC/MS/MS coupled with automated solid-phase extraction sample preparation.

    PubMed

    Tong, Xinchun S; Wang, Junying; Zheng, Song; Pivnichny, James V

    2004-06-29

    Automation of plasma sample preparation for pharmacokinetic studies on VLA-4 antagonists has been achieved by using 96-well format solid-phase extraction operated by Beckman Coulter Biomek 2000 liquid handling system. A Biomek 2000 robot is used to perform fully automated plasma sample preparation tasks that include serial dilution of standard solutions, pipetting plasma samples, addition of standard and internal standard solutions, performing solid-phase extraction (SPE) on Waters OASIS 96-well plates. This automated sample preparation process takes less than 2 h for a typical pharmacokinetic study, including 51 samples, 24 standards, 9 quality controls, and 3-6 dose checks with minimal manual intervention. Extensive validation has been made to ensure the accuracy and reliability of this method. A two-stage vacuum pressure controller has been incorporated in the program to improve SPE efficiency. This automated SPE sample preparation approach combined with liquid chromatography coupled with the high sensitivity and selectivity of tandem mass spectrometry (LC/MS)/MS has been successfully applied on both individual and cassette dosing for pharmacokinetic screening of a large number of VLA-4 antagonists with a limit of quantitation in the range of 1-5 ng/ml. Consequently, a significant throughput increase has been achieved along with an elimination of tedious labor and its consequential tendency to produce errors. Copyright 2004 Elsevier B.V.

  6. EFFECTS OF STORAGE, RNA EXTRACTION, GENECHIP TYPE, AND DONOR SEX ON GENE EXPRESSION PROFILING OF HUMAN WHOLE BLOOD

    EPA Science Inventory

    Background: Gene expression profiling of whole blood may be useful for monitoring toxicological exposure and for diagnosis and monitoring of various diseases. Several methods are available that can be used to transport, store, and extract RNA from whole blood, but it is not clear...

  7. EFFECTS OF STORAGE, RNA EXTRACTION, GENECHIP TYPE, AND DONOR SEX ON GENE EXPRESSION PROFILING OF HUMAN WHOLE BLOOD

    EPA Science Inventory

    Background: Gene expression profiling of whole blood may be useful for monitoring toxicological exposure and for diagnosis and monitoring of various diseases. Several methods are available that can be used to transport, store, and extract RNA from whole blood, but it is not clear...