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Sample records for sensitive multiplex rna

  1. Sensitive, multiplex and direct quantification of RNA sequences using a modified RASL assay

    PubMed Central

    Larman, H. Benjamin; Scott, Erick R.; Wogan, Megan; Oliveira, Glenn; Torkamani, Ali; Schultz, Peter G.

    2014-01-01

    A sensitive and highly multiplex method to directly measure RNA sequence abundance without requiring reverse transcription would be of value for a number of biomedical applications, including high throughput small molecule screening, pathogen transcript detection and quantification of short/degraded RNAs. RNA Annealing, Selection and Ligation (RASL) assays, which are based on RNA template-dependent oligonucleotide probe ligation, have been developed to meet this need, but technical limitations have impeded their adoption. Whereas DNA ligase-based RASL assays suffer from extremely low and sequence-dependent ligation efficiencies that compromise assay robustness, Rnl2 can join a fully DNA donor probe to a 3′-diribonucleotide-terminated acceptor probe with high efficiency on an RNA template strand. Rnl2-based RASL exhibits sub-femtomolar transcript detection sensitivity, and permits the rational tuning of probe signals for optimal analysis by massively parallel DNA sequencing (RASL-seq). A streamlined Rnl2-based RASL-seq protocol was assessed in a small molecule screen using 77 probe sets designed to monitor complex human B cell phenotypes during antibody class switch recombination. Our data demonstrate the robustness, cost-efficiency and broad applicability of Rnl2-based RASL assays. PMID:25063296

  2. A highly multiplexed and sensitive RNA-seq protocol for simultaneous analysis of host and pathogen transcriptomes.

    PubMed

    Avraham, Roi; Haseley, Nathan; Fan, Amy; Bloom-Ackermann, Zohar; Livny, Jonathan; Hung, Deborah T

    2016-08-01

    The ability to simultaneously characterize the bacterial and host expression programs during infection would facilitate a comprehensive understanding of pathogen-host interactions. Although RNA sequencing (RNA-seq) has greatly advanced our ability to study the transcriptomes of prokaryotes and eukaryotes separately, limitations in existing protocols for the generation and analysis of RNA-seq data have hindered simultaneous profiling of host and bacterial pathogen transcripts from the same sample. Here we provide a detailed protocol for simultaneous analysis of host and bacterial transcripts by RNA-seq. Importantly, this protocol details the steps required for efficient host and bacteria lysis, barcoding of samples, technical advances in sample preparation for low-yield sample inputs and a computational pipeline for analysis of both mammalian and microbial reads from mixed host-pathogen RNA-seq data. Sample preparation takes 3 d from cultured cells to pooled libraries. Data analysis takes an additional day. Compared with previous methods, the protocol detailed here provides a sensitive, facile and generalizable approach that is suitable for large-scale studies and will enable the field to obtain in-depth analysis of host-pathogen interactions in infection models. PMID:27442864

  3. Development of Highly Sensitive and Specific mRNA Multiplex System (XCYR1) for Forensic Human Body Fluids and Tissues Identification

    PubMed Central

    Xu, Yan; Xie, Jianhui; Cao, Yu; Zhou, Huaigu; Ping, Yuan; Chen, Liankang; Gu, Lihua; Hu, Wei; Bi, Gang; Ge, Jianye; Chen, Xin; Zhao, Ziqin

    2014-01-01

    The identification of human body fluids or tissues through mRNA-based profiling is very useful for forensic investigations. Previous studies have shown mRNA biomarkers are effective to identify the origin of biological samples. In this study, we selected 16 tissue specific biomarkers to evaluate their specificities and sensitivities for human body fluids and tissues identification, including porphobilinogen deaminase (PBGD), hemoglobin beta (HBB) and Glycophorin A (GLY) for circulatory blood, protamine 2 (PRM2) and transglutaminase 4 (TGM4) for semen, mucin 4 (MUC4) and human beta defensin 1(HBD1) for vaginal secretion, matrix metalloproteinases 7 and 11 (MMP7 and MMP11) for menstrual blood, keratin 4(KRT4) for oral mucosa, loricrin (LOR) and cystatin 6 (CST6) for skin, histatin 3(HTN3) for saliva, statherin (STATH) for nasal secretion, dermcidin (DCD) for sweat and uromodulin (UMOD) for urine. The above mentioned ten common forensic body fluids or tissues were used in the evaluation. Based on the evaluation, a reverse transcription (RT) PCR multiplex assay, XCYR1, which includes 12 biomarkers (i.e., HBB, GLY, HTN3, PRM2, KRT4, MMP11, MUC4, DCD, UMOD, MMP7, TGM4, and STATH) and 2 housekeeping genes [i.e., glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and 18SrRNA], was developed. This assay was further validated with real casework samples and mock samples (with both single source and mixture) and it was approved that XCYR1 is effective to identify common body fluids or tissues (i.e., circulatory blood, saliva, semen, vaginal secretion, menstrual blood, oral mucosa, nasal secretion, sweat and urine) in forensic casework samples. PMID:24991806

  4. Development of highly sensitive and specific mRNA multiplex system (XCYR1) for forensic human body fluids and tissues identification.

    PubMed

    Xu, Yan; Xie, Jianhui; Cao, Yu; Zhou, Huaigu; Ping, Yuan; Chen, Liankang; Gu, Lihua; Hu, Wei; Bi, Gang; Ge, Jianye; Chen, Xin; Zhao, Ziqin

    2014-01-01

    The identification of human body fluids or tissues through mRNA-based profiling is very useful for forensic investigations. Previous studies have shown mRNA biomarkers are effective to identify the origin of biological samples. In this study, we selected 16 tissue specific biomarkers to evaluate their specificities and sensitivities for human body fluids and tissues identification, including porphobilinogen deaminase (PBGD), hemoglobin beta (HBB) and Glycophorin A (GLY) for circulatory blood, protamine 2 (PRM2) and transglutaminase 4 (TGM4) for semen, mucin 4 (MUC4) and human beta defensin 1(HBD1) for vaginal secretion, matrix metalloproteinases 7 and 11 (MMP7 and MMP11) for menstrual blood, keratin 4(KRT4) for oral mucosa, loricrin (LOR) and cystatin 6 (CST6) for skin, histatin 3(HTN3) for saliva, statherin (STATH) for nasal secretion, dermcidin (DCD) for sweat and uromodulin (UMOD) for urine. The above mentioned ten common forensic body fluids or tissues were used in the evaluation. Based on the evaluation, a reverse transcription (RT) PCR multiplex assay, XCYR1, which includes 12 biomarkers (i.e., HBB, GLY, HTN3, PRM2, KRT4, MMP11, MUC4, DCD, UMOD, MMP7, TGM4, and STATH) and 2 housekeeping genes [i.e., glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and 18SrRNA], was developed. This assay was further validated with real casework samples and mock samples (with both single source and mixture) and it was approved that XCYR1 is effective to identify common body fluids or tissues (i.e., circulatory blood, saliva, semen, vaginal secretion, menstrual blood, oral mucosa, nasal secretion, sweat and urine) in forensic casework samples.

  5. Development of highly sensitive and specific mRNA multiplex system (XCYR1) for forensic human body fluids and tissues identification.

    PubMed

    Xu, Yan; Xie, Jianhui; Cao, Yu; Zhou, Huaigu; Ping, Yuan; Chen, Liankang; Gu, Lihua; Hu, Wei; Bi, Gang; Ge, Jianye; Chen, Xin; Zhao, Ziqin

    2014-01-01

    The identification of human body fluids or tissues through mRNA-based profiling is very useful for forensic investigations. Previous studies have shown mRNA biomarkers are effective to identify the origin of biological samples. In this study, we selected 16 tissue specific biomarkers to evaluate their specificities and sensitivities for human body fluids and tissues identification, including porphobilinogen deaminase (PBGD), hemoglobin beta (HBB) and Glycophorin A (GLY) for circulatory blood, protamine 2 (PRM2) and transglutaminase 4 (TGM4) for semen, mucin 4 (MUC4) and human beta defensin 1(HBD1) for vaginal secretion, matrix metalloproteinases 7 and 11 (MMP7 and MMP11) for menstrual blood, keratin 4(KRT4) for oral mucosa, loricrin (LOR) and cystatin 6 (CST6) for skin, histatin 3(HTN3) for saliva, statherin (STATH) for nasal secretion, dermcidin (DCD) for sweat and uromodulin (UMOD) for urine. The above mentioned ten common forensic body fluids or tissues were used in the evaluation. Based on the evaluation, a reverse transcription (RT) PCR multiplex assay, XCYR1, which includes 12 biomarkers (i.e., HBB, GLY, HTN3, PRM2, KRT4, MMP11, MUC4, DCD, UMOD, MMP7, TGM4, and STATH) and 2 housekeeping genes [i.e., glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and 18SrRNA], was developed. This assay was further validated with real casework samples and mock samples (with both single source and mixture) and it was approved that XCYR1 is effective to identify common body fluids or tissues (i.e., circulatory blood, saliva, semen, vaginal secretion, menstrual blood, oral mucosa, nasal secretion, sweat and urine) in forensic casework samples. PMID:24991806

  6. Multiplexed miRNA northern blots via hybridization chain reaction

    PubMed Central

    Schwarzkopf, Maayan; Pierce, Niles A.

    2016-01-01

    Northern blots enable detection of a target RNA of interest in a biological sample using standard benchtop equipment. miRNAs are the most challenging targets as they must be detected with a single short nucleic acid probe. With existing approaches, it is cumbersome to perform multiplexed blots in which several RNAs are detected simultaneously, impeding the study of interacting regulatory elements. Here, we address this shortcoming by demonstrating multiplexed northern blotting based on the mechanism of hybridization chain reaction (HCR). With this approach, nucleic acid probes complementary to RNA targets trigger chain reactions in which fluorophore-labeled DNA hairpins self-assemble into tethered fluorescent amplification polymers. The programmability of HCR allows multiple amplifiers to operate simultaneously and independently within a blot, enabling straightforward multiplexing. We demonstrate simultaneous detection of three endogenous miRNAs in total RNA extracted from 293T and HeLa cells. For a given target, HCR signal scales linearly with target abundance, enabling relative and absolute quantitation. Using non-radioactive HCR, sensitive and selective miRNA detection is achieved using 2′OMe-RNA probes. The HCR northern blot protocol takes ∼1.5 days independent of the number of target RNAs. PMID:27270083

  7. Multiplexed miRNA northern blots via hybridization chain reaction.

    PubMed

    Schwarzkopf, Maayan; Pierce, Niles A

    2016-09-01

    Northern blots enable detection of a target RNA of interest in a biological sample using standard benchtop equipment. miRNAs are the most challenging targets as they must be detected with a single short nucleic acid probe. With existing approaches, it is cumbersome to perform multiplexed blots in which several RNAs are detected simultaneously, impeding the study of interacting regulatory elements. Here, we address this shortcoming by demonstrating multiplexed northern blotting based on the mechanism of hybridization chain reaction (HCR). With this approach, nucleic acid probes complementary to RNA targets trigger chain reactions in which fluorophore-labeled DNA hairpins self-assemble into tethered fluorescent amplification polymers. The programmability of HCR allows multiple amplifiers to operate simultaneously and independently within a blot, enabling straightforward multiplexing. We demonstrate simultaneous detection of three endogenous miRNAs in total RNA extracted from 293T and HeLa cells. For a given target, HCR signal scales linearly with target abundance, enabling relative and absolute quantitation. Using non-radioactive HCR, sensitive and selective miRNA detection is achieved using 2'OMe-RNA probes. The HCR northern blot protocol takes ∼1.5 days independent of the number of target RNAs. PMID:27270083

  8. Quantifying RNA allelic ratios by microfluidic multiplex PCR and sequencing.

    PubMed

    Zhang, Rui; Li, Xin; Ramaswami, Gokul; Smith, Kevin S; Turecki, Gustavo; Montgomery, Stephen B; Li, Jin Billy

    2014-01-01

    We developed a targeted RNA sequencing method that couples microfluidics-based multiplex PCR and deep sequencing (mmPCR-seq) to uniformly and simultaneously amplify up to 960 loci in 48 samples independently of their gene expression levels and to accurately and cost-effectively measure allelic ratios even for low-quantity or low-quality RNA samples. We applied mmPCR-seq to RNA editing and allele-specific expression studies. mmPCR-seq complements RNA-seq for studying allelic variations in the transcriptome.

  9. Assessment of microRNA differential expression and detection in multiplexed small RNA sequencing data.

    PubMed

    Campbell, Joshua D; Liu, Gang; Luo, Lingqi; Xiao, Ji; Gerrein, Joseph; Juan-Guardela, Brenda; Tedrow, John; Alekseyev, Yuriy O; Yang, Ivana V; Correll, Mick; Geraci, Mark; Quackenbush, John; Sciurba, Frank; Schwartz, David A; Kaminski, Naftali; Johnson, W Evan; Monti, Stefano; Spira, Avrum; Beane, Jennifer; Lenburg, Marc E

    2015-02-01

    Small RNA sequencing can be used to gain an unprecedented amount of detail into the microRNA transcriptome. The relatively high cost and low throughput of sequencing bases technologies can potentially be offset by the use of multiplexing. However, multiplexing involves a trade-off between increased number of sequenced samples and reduced number of reads per sample (i.e., lower depth of coverage). To assess the effect of different sequencing depths owing to multiplexing on microRNA differential expression and detection, we sequenced the small RNA of lung tissue samples collected in a clinical setting by multiplexing one, three, six, nine, or 12 samples per lane using the Illumina HiSeq 2000. As expected, the numbers of reads obtained per sample decreased as the number of samples in a multiplex increased. Furthermore, after normalization, replicate samples included in distinct multiplexes were highly correlated (R > 0.97). When detecting differential microRNA expression between groups of samples, microRNAs with average expression >1 reads per million (RPM) had reproducible fold change estimates (signal to noise) independent of the degree of multiplexing. The number of microRNAs detected was strongly correlated with the log2 number of reads aligning to microRNA loci (R = 0.96). However, most additional microRNAs detected in samples with greater sequencing depth were in the range of expression which had lower fold change reproducibility. These findings elucidate the trade-off between increasing the number of samples in a multiplex with decreasing sequencing depth and will aid in the design of large-scale clinical studies exploring microRNA expression and its role in disease.

  10. A protein multiplex microarray substrate with high sensitivity and specificity

    PubMed Central

    Fici, Dolores A.; McCormick, William; Brown, David W.; Herrmann, John E.; Kumar, Vikram; Awdeh, Zuheir L.

    2010-01-01

    The problems that have been associated with protein multiplex microarray immunoassay substrates and existing technology platforms include: binding, sensitivity, a low signal to noise ratio, target immobilization and the optimal simultaneous detection of diverse protein targets. Current commercial substrates for planar multiplex microarrays rely on protein attachment chemistries that range from covalent attachment to affinity ligand capture, to simple adsorption. In this pilot study, experimental performance parameters for direct monoclonal mouse IgG detection were compared for available two and three dimensional slide surface coatings with a new colloidal nitrocellulose substrate. New technology multiplex microarrays were also developed and evaluated for the detection of pathogen specific antibodies in human serum and the direct detection of enteric viral antigens. Data supports the nitrocellulose colloid as an effective reagent with the capacity to immobilize sufficient diverse protein target quantities for increased specificory signal without compromising authentic protein structure. The nitrocellulose colloid reagent is compatible with the array spotters and scanners routinely used for microarray preparation and processing. More importantly, as an alternate to fluorescence, colorimetric chemistries may be used for specific and sensitive protein target detection. The advantages of the nitrocellulose colloid platform indicate that this technology may be a valuable tool for the further development and expansion of multiplex microarray immunoassays in both the clinical and research laborat environment. PMID:20974147

  11. RNA Imaging with Multiplexed Error Robust Fluorescence in situ Hybridization

    PubMed Central

    Moffitt, Jeffrey R.; Zhuang, Xiaowei

    2016-01-01

    Quantitative measurements of both the copy number and spatial distribution of large fractions of the transcriptome in single-cells could revolutionize our understanding of a variety of cellular and tissue behaviors in both healthy and diseased states. Single-molecule Fluorescence In Situ Hybridization (smFISH)—an approach where individual RNAs are labeled with fluorescent probes and imaged in their native cellular and tissue context—provides both the copy number and spatial context of RNAs but has been limited in the number of RNA species that can be measured simultaneously. Here we describe Multiplexed Error Robust Fluorescence In Situ Hybridization (MERFISH), a massively parallelized form of smFISH that can image and identify hundreds to thousands of different RNA species simultaneously with high accuracy in individual cells in their native spatial context. We provide detailed protocols on all aspects of MERFISH, including probe design, data collection, and data analysis to allow interested laboratories to perform MERFISH measurements themselves. PMID:27241748

  12. Wavelength-multiplexing phase-sensitive surface plasmon imaging sensor.

    PubMed

    Shao, Yonghong; Li, Yan; Gu, Dayong; Zhang, Kai; Qu, Junle; He, Jianan; Li, Xuejin; Wu, Shu-Yuen; Ho, Ho-Pui; Somekh, Michael G; Niu, Hanben

    2013-05-01

    A wavelength-multiplexing phase-sensitive surface plasmon resonance (SPR) imaging sensor offering wide dynamic detection range and microarray capability is reported. Phase detection is accomplished by performing self-interference between the s- and p- polarizations within the signal beam. A liquid crystal tunable filter is used to sequentially select the SPR excitation wavelength from a white light source. This wavelength-multiplexing approach enables fast detection of the sensor's SPR phase response over a wide range of wavelengths, thereby covering literally any regions of interest within the SPR dip and thus maintaining the highest sensitivity point at all times. The phase-sensitive approach is particularly important for imaging SPR sensing applications because of its less stringent requirements for intensity signal-to-noise ratio, which also means the possibility of using uncooled modest resolution analog-to-digital conversion imaging devices. Experimental results demonstrate a resolution of 2.7×10(-7) RIU with a dynamic range of 0.0138 RIU. PMID:23632487

  13. Rapid and multiplex microRNA detection on graphically encoded silica suspension array.

    PubMed

    Jiang, Li; Shen, Ye; Zheng, Kexiao; Li, Jiong

    2014-11-15

    MicroRNA (miRNA), an 18-24-nucleotide noncoding RNA molecule, has become an ideal class of biomarker candidates for clinical diagnosis of cancers. By now, a number of detection methods for miRNAs have been developed on planar arrays and suspension arrays. In this work, we describe a hybridization-triggered fluorescence strategy for label-free and multiplex miRNA detection on graphically encoded silica suspension array. The total RNA is directly applied for analysis with an 8-mer Universal Tag which can be selectively captured by the capture probe via base-stacking effects. Benefiting from base-stacking effects, this novel method exhibits superb discrimination ability toward the 5' and 3' end single-nucleotide alteration. Mature miRNAs can be distinguished from their corresponding pre-miRNAs easily. Moreover, the estimated detection limit of 5 amol is comparable to some of the most sensitive methods. All these mentioned characteristics offer exciting possibilities for discovery and clinical applications.

  14. Developed and evaluated a multiplex mRNA profiling system for body fluid identification in Chinese Han population.

    PubMed

    Song, Feng; Luo, Haibo; Hou, Yiping

    2015-10-01

    In forensic casework, identification the cellular origin from a biological sample is crucial to the case investigation and reconstruction in crime scene. DNA/RNA co-extraction for STR typing and human body fluids identification has been proposed as an efficient and comprehensive assay for forensic analysis. Several cell-specific messenger RNA (mRNA) markers for identification of the body fluids have been proposed by previous studies. In this study, a novel multiplex mRNA profiling system included 19 markers was developed and performed by reverse transcription endpoint polymerase chain reaction (RT-PCR). The multiplex combined 3 housekeeping gene markers and 16 cell-specific markers that have been used to identify five types of human body fluids: peripheral blood, semen, saliva, vaginal secretions and menstrual blood. The specificity, sensitivity, stability and detectability of the mixture were explored in our study. Majority of the cell-specific mRNA markers showed high specificity, although cross-reactivity was observed sporadically. Specific profiling for per body fluid was obtained. Moreover, the interpretation guidelines for inference of body fluid types were performed according to the A. Lindenbergh et al. The scoring guidelines can be applied to any RNA multiplex, which was based on six different scoring categories (observed, observed and fits, sporadically observed and fits, not observed, sporadically observed, not reliable, and non-specific due to high input). The simultaneous extraction of DNA showed positive full or partial profiling results of all samples. It demonstrated that the approach of combined STR-profiling and RNA profiling was suitable and reliable to detect the donor and origin of human body fluids in Chinese Han population. PMID:26311108

  15. Developed and evaluated a multiplex mRNA profiling system for body fluid identification in Chinese Han population.

    PubMed

    Song, Feng; Luo, Haibo; Hou, Yiping

    2015-10-01

    In forensic casework, identification the cellular origin from a biological sample is crucial to the case investigation and reconstruction in crime scene. DNA/RNA co-extraction for STR typing and human body fluids identification has been proposed as an efficient and comprehensive assay for forensic analysis. Several cell-specific messenger RNA (mRNA) markers for identification of the body fluids have been proposed by previous studies. In this study, a novel multiplex mRNA profiling system included 19 markers was developed and performed by reverse transcription endpoint polymerase chain reaction (RT-PCR). The multiplex combined 3 housekeeping gene markers and 16 cell-specific markers that have been used to identify five types of human body fluids: peripheral blood, semen, saliva, vaginal secretions and menstrual blood. The specificity, sensitivity, stability and detectability of the mixture were explored in our study. Majority of the cell-specific mRNA markers showed high specificity, although cross-reactivity was observed sporadically. Specific profiling for per body fluid was obtained. Moreover, the interpretation guidelines for inference of body fluid types were performed according to the A. Lindenbergh et al. The scoring guidelines can be applied to any RNA multiplex, which was based on six different scoring categories (observed, observed and fits, sporadically observed and fits, not observed, sporadically observed, not reliable, and non-specific due to high input). The simultaneous extraction of DNA showed positive full or partial profiling results of all samples. It demonstrated that the approach of combined STR-profiling and RNA profiling was suitable and reliable to detect the donor and origin of human body fluids in Chinese Han population.

  16. An improved method for absolute quantification of mRNA using multiplex polymerase chain reaction: determination of renin and angiotensinogen mRNA levels in various tissues.

    PubMed

    Dostal, D E; Rothblum, K N; Baker, K M

    1994-12-01

    We have developed a multiplex, competitive, reverse-transcriptase polymerase chain reaction (RT-PCR) method which measures absolute levels of renin, angiotensinogen, and the housekeeping transcript elongation factor-1 alpha (EF-1 alpha) mRNA. Sample RNA was simultaneously titrated with serial dilutions of renin, angiotensinogen, and EF-1 alpha competitor RNAs which flanked the endogenous concentrations of target transcripts. The samples were coreverse transcribed in the presence of random primers and resulting first-strand cDNA was coamplified for 10-15 cycles with [32P]-dCTP and primers for renin angiotensinogen, after which EF-1 alpha primers were added. Amplified DNA was separated by electrophoresis on polyacrylamide gel and radioactivity in the bands was quantified by direct radioanalytical scanning. Three conditions were necessary to obtain absolute quantification of renin and angiotensinogen mRNA levels: (a) exogenous competitor RNA was used to control for tube-to-tube variability in the efficiencies of reverse transcription and amplification; (b) Sample RNA was titrated with flanking concentrations of competitor RNA to correct for intraassay differences in the efficiency of amplification due to concentration differences between competitor and target templates; and (c) a housekeeping transcript EF-1 alpha was used to control for tube-to-tube differences in RNA loading and/or degradation. We show that the multiplex RT-PCR method is precise and accurate over approximately three logs of transcript concentration and sensitive to less than 5 and 0.5 fg for renin and angiotensinogen mRNA, respectively. This method will be useful for absolute quantification of target mRNAs, especially when the amount of sample RNA is limited or unknown and/or the gene expression is low. PMID:7887470

  17. A nucleic acid strand displacement system for the multiplexed detection of tuberculosis-specific mRNA using quantum dots

    NASA Astrophysics Data System (ADS)

    Gliddon, H. D.; Howes, P. D.; Kaforou, M.; Levin, M.; Stevens, M. M.

    2016-05-01

    The development of rapid, robust and high performance point-of-care diagnostics relies on the advancement and combination of various areas of research. We have developed an assay for the detection of multiple mRNA molecules that combines DNA nanotechnology with fluorescent nanomaterials. The core switching mechanism is toehold-mediated strand displacement. We have used fluorescent quantum dots (QDs) as signal transducers in this assay, as they bring many benefits including bright fluorescence and multiplexing abilities. The resulting assay is capable of multiplexed detection of long RNA targets against a high concentration of background non-target RNA, with high sensitivity and specificity and limits of detection in the nanomolar range using only a standard laboratory plate reader. We demonstrate the utility of our QD-based system for the detection of two genes selected from a microarray-derived tuberculosis-specific gene expression signature. Levels of up- and downregulated gene transcripts comprising this signature can be combined to give a disease risk score, making the signature more amenable for use as a diagnostic marker. Our QD-based approach to detect these transcripts could pave the way for novel diagnostic assays for tuberculosis.The development of rapid, robust and high performance point-of-care diagnostics relies on the advancement and combination of various areas of research. We have developed an assay for the detection of multiple mRNA molecules that combines DNA nanotechnology with fluorescent nanomaterials. The core switching mechanism is toehold-mediated strand displacement. We have used fluorescent quantum dots (QDs) as signal transducers in this assay, as they bring many benefits including bright fluorescence and multiplexing abilities. The resulting assay is capable of multiplexed detection of long RNA targets against a high concentration of background non-target RNA, with high sensitivity and specificity and limits of detection in the nanomolar

  18. Highly sensitive and multiplexed platforms for allergy diagnostics

    NASA Astrophysics Data System (ADS)

    Monroe, Margo R.

    Allergy is a disorder of the immune system caused by an immune response to otherwise harmless environmental allergens. Currently 20% of the US population is allergic and 90% of pediatric patients and 60% of adult patients with asthma have allergies. These percentages have increased by 18.5% in the past decade, with predicted similar trends for the future. Here we design sensitive, multiplexed platforms to detect allergen-specific IgE using the Interferometric Reflectance Imaging Sensor (IRIS) for various clinical settings. A microarray platform for allergy diagnosis allows for testing of specific IgE sensitivity to a multitude of allergens, while requiring only small volumes of patient blood sample. However, conventional fluorescent microarray technology is limited by i) the variation of probe immobilization, which hinders the ability to make quantitative, assertive, and statistically relevant conclusions necessary in immunodiagnostics and ii) the use of fluorophore labels, which is not suitable for some clinical applications due to the tendency of fluorophores to stick to blood particulates and require daily calibration methods. This calibrated fluorescence enhancement (CaFE) method integrates the low magnification modality of IRIS with enhanced fluorescence sensing in order to directly correlate immobilized probe (major allergens) density to allergen-specific IgE in patient serum. However, this platform only operates in processed serum samples, which is not ideal for point of care testing. Thus, a high magnification modality of IRIS was adapted as an alternative allergy diagnostic platform to automatically discriminate and size single nanoparticles bound to specific IgE in unprocessed, characterized human blood and serum samples. These features make IRIS an ideal candidate for clinical and diagnostic applications, such a POC testing. The high magnification (nanoparticle counting) modality in conjunction with low magnification of IRIS in a combined instrument

  19. Multiplexed miRNA Fluorescence In Situ Hybridization for Formalin-Fixed Paraffin-Embedded Tissues

    PubMed Central

    Renwick, Neil; Cekan, Pavol; Bognanni, Claudia; Tuschl, Thomas

    2015-01-01

    Multiplexed miRNA fluorescence in situ hybridization (miRNA FISH) is an advanced method for visualizing differentially expressed miRNAs, together with other reference RNAs, in archival tissues. Some miRNAs are excellent disease biomarkers due to their abundance and cell-type specificity. However, these short RNA molecules are difficult to visualize due to loss by diffusion, probe mishybridization, and signal detection and signal amplification issues. Here, we describe a reliable and adjustable method for visualizing and normalizing miRNA signals in formalin-fixed paraffin-embedded (FFPE) tissue sections. PMID:25218385

  20. Development and evaluation of multiplex RT-LAMP assays for rapid and sensitive detection of foot-and-mouth disease virus.

    PubMed

    Yamazaki, Wataru; Mioulet, Valérie; Murray, Lee; Madi, Mikidache; Haga, Takeshi; Misawa, Naoaki; Horii, Yoichiro; King, Donald P

    2013-09-01

    This paper describes the evaluation of four novel real-time multiplex reverse transcription loop-mediated isothermal amplification (RT-LAMP) assays for rapid and sensitive diagnosis of foot-and-mouth disease (FMD). In order to overcome the genetic diversity of FMD viruses (FMDV), these multiplex RT-LAMP assay pairs were established by combining four newly designed primer sets with two primer sets that had been previously published. Using a real-time turbidimeter to detect amplification products and a panel of 300 samples collected throughout the world over a 78-year period, the performance of the multiplex RT-LAMP assays was compared with a FMDV-specific real-time RT-PCR assay. The most successful of the four multiplex RT-LAMP assays achieved a diagnostic sensitivity and specificity of 98.0% and 98.1%, and did not falsely detect FMDV in known negatives or samples containing swine vesicular disease virus, vesicular stomatitis virus or vesicular exanthema of swine virus. Furthermore, the analytical sensitivity of this multiplex RT-LAMP assay was at least as good as the individual component RT-LAMP tests. This is the first report of the development of a multiplex RT-LAMP to accommodate the high sequence variability encountered in RNA virus genomes and these results support the use of RT-LAMP as a cost-effective tool for simple diagnosis of FMD. PMID:23583488

  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. miRge - A Multiplexed Method of Processing Small RNA-Seq Data to Determine MicroRNA Entropy

    PubMed Central

    Myers, Jason R.; Gupta, Simone; Weng, Lien-Chun; Ashton, John M.; Cornish, Toby C.; Pandey, Akhilesh; Halushka, Marc K.

    2015-01-01

    Small RNA RNA-seq for microRNAs (miRNAs) is a rapidly developing field where opportunities still exist to create better bioinformatics tools to process these large datasets and generate new, useful analyses. We built miRge to be a fast, smart small RNA-seq solution to process samples in a highly multiplexed fashion. miRge employs a Bayesian alignment approach, whereby reads are sequentially aligned against customized mature miRNA, hairpin miRNA, noncoding RNA and mRNA sequence libraries. miRNAs are summarized at the level of raw reads in addition to reads per million (RPM). Reads for all other RNA species (tRNA, rRNA, snoRNA, mRNA) are provided, which is useful for identifying potential contaminants and optimizing small RNA purification strategies. miRge was designed to optimally identify miRNA isomiRs and employs an entropy based statistical measurement to identify differential production of isomiRs. This allowed us to identify decreasing entropy in isomiRs as stem cells mature into retinal pigment epithelial cells. Conversely, we show that pancreatic tumor miRNAs have similar entropy to matched normal pancreatic tissues. In a head-to-head comparison with other miRNA analysis tools (miRExpress 2.0, sRNAbench, omiRAs, miRDeep2, Chimira, UEA small RNA Workbench), miRge was faster (4 to 32-fold) and was among the top-two methods in maximally aligning miRNAs reads per sample. Moreover, miRge has no inherent limits to its multiplexing. miRge was capable of simultaneously analyzing 100 small RNA-Seq samples in 52 minutes, providing an integrated analysis of miRNA expression across all samples. As miRge was designed for analysis of single as well as multiple samples, miRge is an ideal tool for high and low-throughput users. miRge is freely available at http://atlas.pathology.jhu.edu/baras/miRge.html. PMID:26571139

  3. A multiplex (m)RNA-profiling system for the forensic identification of body fluids and contact traces.

    PubMed

    Lindenbergh, Alexander; de Pagter, Mirjam; Ramdayal, Geeta; Visser, Mijke; Zubakov, Dmitry; Kayser, Manfred; Sijen, Titia

    2012-09-01

    In current forensic practice, information about the possible biological origin of forensic traces is mostly determined using protein-based presumptive testing. Recently, messenger RNA-profiling has emerged as an alternative strategy to examine the biological origin. Here we describe the development of a single multiplex mRNA-based system for the discrimination of the most common forensic body fluids as well as skin cells. A DNA/RNA co-isolation protocol was established that results in DNA yields equivalent to our standard in-house validated DNA extraction procedure which uses silica-based columns. An endpoint RT-PCR assay was developed that simultaneously amplifies 19 (m)RNA markers. This multiplex assay analyses three housekeeping, three blood, two saliva, two semen, two menstrual secretion, two vaginal mucosa, three general mucosa and two skin markers. The assay has good sensitivity as full RNA profiles for blood, semen and saliva were obtained when using ≥0.05 μL body fluid starting material whereas full DNA profiles were obtained with ≥0.1 μL. We investigated the specificity of the markers by analysing 15 different sets of each type of body fluid and skin with each set consisting of 8 individuals. Since skin markers have not been incorporated in multiplex endpoint PCR assays previously, we analysed these markers in more detail. Interestingly, both skin markers gave a positive result in samplings of the hands, feet, back and lips but negative in tongue samplings. Positive identification (regarding both DNA- and RNA-profiling) was obtained for specimens stored for many years, e.g. blood (28 years-old), semen (28 years-old), saliva (6 years-old), skin (10 years-old) and menstrual secretion (4 years-old). The described approach of combined DNA- and RNA-profiling of body fluids and contact traces assists in the interpretation of forensic stains by providing information about not only the donor(s) that contributed to the stain but also by indicating which cell

  4. A multiplex (m)RNA-profiling system for the forensic identification of body fluids and contact traces.

    PubMed

    Lindenbergh, Alexander; de Pagter, Mirjam; Ramdayal, Geeta; Visser, Mijke; Zubakov, Dmitry; Kayser, Manfred; Sijen, Titia

    2012-09-01

    In current forensic practice, information about the possible biological origin of forensic traces is mostly determined using protein-based presumptive testing. Recently, messenger RNA-profiling has emerged as an alternative strategy to examine the biological origin. Here we describe the development of a single multiplex mRNA-based system for the discrimination of the most common forensic body fluids as well as skin cells. A DNA/RNA co-isolation protocol was established that results in DNA yields equivalent to our standard in-house validated DNA extraction procedure which uses silica-based columns. An endpoint RT-PCR assay was developed that simultaneously amplifies 19 (m)RNA markers. This multiplex assay analyses three housekeeping, three blood, two saliva, two semen, two menstrual secretion, two vaginal mucosa, three general mucosa and two skin markers. The assay has good sensitivity as full RNA profiles for blood, semen and saliva were obtained when using ≥0.05 μL body fluid starting material whereas full DNA profiles were obtained with ≥0.1 μL. We investigated the specificity of the markers by analysing 15 different sets of each type of body fluid and skin with each set consisting of 8 individuals. Since skin markers have not been incorporated in multiplex endpoint PCR assays previously, we analysed these markers in more detail. Interestingly, both skin markers gave a positive result in samplings of the hands, feet, back and lips but negative in tongue samplings. Positive identification (regarding both DNA- and RNA-profiling) was obtained for specimens stored for many years, e.g. blood (28 years-old), semen (28 years-old), saliva (6 years-old), skin (10 years-old) and menstrual secretion (4 years-old). The described approach of combined DNA- and RNA-profiling of body fluids and contact traces assists in the interpretation of forensic stains by providing information about not only the donor(s) that contributed to the stain but also by indicating which cell

  5. The development of a mRNA multiplex RT-PCR assay for the definitive identification of body fluids.

    PubMed

    Fleming, Rachel I; Harbison, SallyAnn

    2010-07-01

    With current methodology, DNA profiling can identify an individual from a sample of biological material but it does not reveal what body fluid or tissue source the DNA profile originated from. We have developed a multiplex PCR system using messenger RNA (mRNA) that can identify blood, saliva, semen and menstrual blood in individual stains or in mixtures of body fluids. Messenger RNA transcripts specific to each type of body fluid have been identified and a multiplex reverse transcriptase-polymerase chain reaction (RT-PCR) system developed to identify these body fluids along with three housekeeping genes. This multiplex can detect semen and seminal fluid (semen without spermatozoa present). Furthermore, we have targeted the co-isolation of RNA and DNA from the same sample and, with the RT-PCR multiplex, we can determine the type of body fluid present as well as generate a DNA profile(s) from the same stain.

  6. The development of a mRNA multiplex RT-PCR assay for the definitive identification of body fluids.

    PubMed

    Fleming, Rachel I; Harbison, SallyAnn

    2010-07-01

    With current methodology, DNA profiling can identify an individual from a sample of biological material but it does not reveal what body fluid or tissue source the DNA profile originated from. We have developed a multiplex PCR system using messenger RNA (mRNA) that can identify blood, saliva, semen and menstrual blood in individual stains or in mixtures of body fluids. Messenger RNA transcripts specific to each type of body fluid have been identified and a multiplex reverse transcriptase-polymerase chain reaction (RT-PCR) system developed to identify these body fluids along with three housekeeping genes. This multiplex can detect semen and seminal fluid (semen without spermatozoa present). Furthermore, we have targeted the co-isolation of RNA and DNA from the same sample and, with the RT-PCR multiplex, we can determine the type of body fluid present as well as generate a DNA profile(s) from the same stain. PMID:20457026

  7. Terbium complex to quantum dot Förster resonance energy transfer for homogeneous and multiplexed microRNA assay (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Qiu, Xue; Hildebrandt, Niko

    2016-03-01

    The importance of microRNA (miRNA) dysregulation in the development and progression of diseases has made these short-length nucleic acids to next generation biomarkers. Tb-to-QD Förster resonance energy transfer (FRET) has several unique advantages over organic dye-based FRET systems for biomolecular sensing. Large Förster distances (6-11 nm) offer much high FRET efficiencies, exceptionally long Tb excited-state lifetimes (ms) enable time-gated detection void of autofluorecence background, and the narrow, symmetric, and tunable emission bands of QDs provide unrivaled potential for multiplexing. Here we report a rapid and homogeneous method to sensitively detect three different miRNAs (hsa-miR-20a-5p, hsa-miR-20b-5p, and hsa-miR-21-5p) from a single 150 µL sample based on multiplexed FRET between a luminescent Lumi4-Tb complex and three different QDs. The biosensing approach exploits both base pairing and stacking. Careful design and optimization of sequence lengths and orientations of the QD and Tb-DNA conjugates was performed to provide maximum selectivity and sensitivity for all three miRNA biomarkers. The assays work at room temperature and were designed for their application on a KRYPTOR diagnostic plate reader system.Only 30 min of sample incubation and 7.5 s of measurement are required to obtain ca. 1 nM (subpicomol) detection limits. We also demonstrate precise multiplexed measurements of these miRNAs at different and varying concentrations and the feasibility of adapting the technology to point-of-care testing (POCT) in buffer containing 10% serum. Our assay does not only demonstrate an important milestone for the integration of quantum dots to multiplexed clinical diagnostics but also a unique rapid miRNA detection technology that is complimentary to the rather complicated high-throughput and high-sensitivity approaches that are established today.

  8. Sensitivity of mRNA Translation

    PubMed Central

    Poker, Gilad; Margaliot, Michael; Tuller, Tamir

    2015-01-01

    Using the dynamic mean-field approximation of the totally asymmetric simple exclusion process (TASEP), we investigate the effect of small changes in the initiation, elongation, and termination rates along the mRNA strand on the steady-state protein translation rate. We show that the sensitivity of mRNA translation is equal to the sensitivity of the maximal eigenvalue of a symmetric, nonnegative, tridiagonal, and irreducible matrix. This leads to new analytical results as well as efficient numerical schemes that are applicable for large-scale models. Our results show that in the usual endogenous case, when initiation is more rate-limiting than elongation, the sensitivity of the translation rate to small mutations rapidly increases towards the 5′ end of the ORF. When the initiation rate is high, as may be the case for highly expressed and/or heterologous optimized genes, the maximal sensitivity is with respect to the elongation rates at the middle of the mRNA strand. We also show that the maximal possible effect of a small increase/decrease in any of the rates along the mRNA is an increase/decrease of the same magnitude in the translation rate. These results are in agreement with previous molecular evolutionary and synthetic biology experimental studies. PMID:26238363

  9. Multiplex qRT-PCR for the Detection of Western Equine Encephalomyelitis, St. Louis Encephalitis, and West Nile Viral RNA in Mosquito Pools (Diptera: Culicidae).

    PubMed

    Brault, Aaron C; Fang, Ying; Reisen, William K

    2015-05-01

    Following the introduction of West Nile virus into California during the summer of 2003, public health and vector control programs expanded surveillance efforts and were in need of diagnostics capable of rapid, sensitive, and specific detection of arbovirus infections of mosquitoes to inform decision support for intervention. Development of a multiplex TaqMan or real-time semiquantitative reverse transcription polymerase chain reaction (RT-PCR) assay in which three virus specific primer-probe sets were used in the same reaction is described herein for the detection of western equine encephalomyelitis, St. Louis encephalitis and West Nile viral RNA. Laboratory validation and field data from 10 transmission seasons are reported. The comparative sensitivity and specificity of this multiplex assay to singleplex RT-PCR as well as an antigen detection (rapid analyte measurement platform) and standard plaque assays indicate this assay to be rapid and useful in providing mosquito infection data to estimate outbreak risk. PMID:26334826

  10. Multiplex qRT-PCR for the Detection of Western Equine Encephalomyelitis, St. Louis Encephalitis, and West Nile Viral RNA in Mosquito Pools (Diptera: Culicidae).

    PubMed

    Brault, Aaron C; Fang, Ying; Reisen, William K

    2015-05-01

    Following the introduction of West Nile virus into California during the summer of 2003, public health and vector control programs expanded surveillance efforts and were in need of diagnostics capable of rapid, sensitive, and specific detection of arbovirus infections of mosquitoes to inform decision support for intervention. Development of a multiplex TaqMan or real-time semiquantitative reverse transcription polymerase chain reaction (RT-PCR) assay in which three virus specific primer-probe sets were used in the same reaction is described herein for the detection of western equine encephalomyelitis, St. Louis encephalitis and West Nile viral RNA. Laboratory validation and field data from 10 transmission seasons are reported. The comparative sensitivity and specificity of this multiplex assay to singleplex RT-PCR as well as an antigen detection (rapid analyte measurement platform) and standard plaque assays indicate this assay to be rapid and useful in providing mosquito infection data to estimate outbreak risk.

  11. Nanogold bioconjugates for direct and sensitive multiplexed immunosensing.

    PubMed

    Dobosz, P; Morais, S; Puchades, R; Maquieira, A

    2015-07-15

    The use of nanogold bioconjugates for direct detection of the antibody-antigen immunoreaction is addressed. The integration of gold nanoparticles tracers as signal generators in microarray immunosensing and compact disc detection technique show important advantages to reach sensitive, selective, high throughput, reliable and cost-effective assays. For that, a thorough study of the performances of the size of spherical nanogold particles and coating density was developed. The size of the nanoparticle determines the optimal antibody dilution, being the smaller particles the best performing ones. Enhancement effect of lower size is also studied. The gold labeling method do not affects the recognition capability of the labeled proteins. As a proof of concept, the nanoconjugates were used for the simultaneous and direct determination of small molecules. Employing nanogold bioconjugates as recognition labels resulted in robust and reliable assays, reaching a sensitivity of 0.03 and 1.3μg/L for sulfasalazine and atrazine, respectively. This shows that the use of nanogold bioconjugates for direct immunosensing is very competitive, achieving highly sensitive and reproducible assays (RSD<10%). This approach would simultaneously determine both small and large molecular size targets, in different formats, using the same detection mode what paves the way for many other applications in different scenarios.

  12. Multiplex mRNA profiling for the identification of body fluids.

    PubMed

    Juusola, Jane; Ballantyne, Jack

    2005-08-11

    We report the development of a multiplex reverse transcription-polymerase chain reaction (RT-PCR) method for the definitive identification of the body fluids that are commonly encountered in forensic casework analysis, namely blood, saliva, semen, and vaginal secretions. Using selected genes that we have identified as being expressed in a tissue-specific manner we have developed a multiplex RT-PCR assay which is composed of eight body fluid-specific genes and that is optimized for the detection of blood, saliva, semen, and vaginal secretions as single or mixed stains. The genes include beta-spectrin (SPTB) and porphobilinogen deaminase (PBGD) for blood, statherin (STATH) and histatin 3 (HTN3) for saliva, protamine 1 (PRM1) and protamine 2 (PRM2) for semen, and human beta-defensin 1 (HBD-1) and mucin 4 (MUC4) for vaginal secretions. The known or presumed functions of these genes suggest an extremely restricted pattern of gene expression, which is a basic requirement for incorporation into a tissue-specific assay. The methodology is based upon gene expression profiling analysis in which the body fluid-specific genes are identified by detecting the presence of appropriate mRNA species using capillary electrophoresis/laser induced fluorescence. An mRNA-based approach, such as the multiplex RT-PCR method described in the present work, allows for the facile identification of the tissue components present in a body fluid stain and could supplant the battery of serological and biochemical tests currently employed in the forensic serology laboratory.

  13. Extensible Multiplex Real-time PCR of MicroRNA Using Microparticles

    PubMed Central

    Jung, Seungwon; Kim, Junsun; Lee, Dong Jin; Oh, Eun Hae; Lim, Hwasup; Kim, Kwang Pyo; Choi, Nakwon; Kim, Tae Song; Kim, Sang Kyung

    2016-01-01

    Multiplex quantitative real-time PCR (qPCR), which measures multiple DNAs in a given sample, has received significant attention as a mean of verifying the rapidly increasing genetic targets of interest in single phenotype. Here we suggest a readily extensible qPCR for the expression analysis of multiple microRNA (miRNA) targets using microparticles of primer-immobilized networks as discrete reactors. Individual particles, 200~500 μm in diameter, are identified by two-dimensional codes engraved into the particles and the non-fluorescent encoding allows high-fidelity acquisition of signal in real-time PCR. During the course of PCR, the amplicons accumulate in the volume of the particles with high reliability and amplification efficiency over 95%. In a quick assay comprising of tens of particles holding different primers, each particle brings the independent real-time amplification curve representing the quantitative information of each target. Limited amount of sample was analyzed simultaneously in single chamber through this highly multiplexed qPCR; 10 kinds of miRNAs from purified extracellular vesicles (EVs). PMID:26964639

  14. Visualization and tracking of tumour extracellular vesicle delivery and RNA translation using multiplexed reporters

    PubMed Central

    Lai, Charles P.; Kim, Edward Y.; Badr, Christian E.; Weissleder, Ralph; Mempel, Thorsten R.; Tannous, Bakhos A.; Breakefield, Xandra O.

    2015-01-01

    Accurate spatiotemporal assessment of extracellular vesicle (EV) delivery and cargo RNA translation requires specific and robust live-cell imaging technologies. Here we engineer optical reporters to label multiple EV populations for visualization and tracking of tumour EV release, uptake and exchange between cell populations both in culture and in vivo. Enhanced green fluorescence protein (EGFP) and tandem dimer Tomato (tdTomato) were fused at NH2-termini with a palmitoylation signal (PalmGFP, PalmtdTomato) for EV membrane labelling. To monitor EV-RNA cargo, transcripts encoding PalmtdTomato were tagged with MS2 RNA binding sequences and detected by co-expression of bacteriophage MS2 coat protein fused with EGFP. By multiplexing fluorescent and bioluminescent EV membrane reporters, we reveal the rapid dynamics of both EV uptake and translation of EV-delivered cargo mRNAs in cancer cells that occurred within 1-hour post-horizontal transfer between cells. These studies confirm that EV-mediated communication is dynamic and multidirectional between cells with delivery of functional mRNA. PMID:25967391

  15. Visualization and tracking of tumour extracellular vesicle delivery and RNA translation using multiplexed reporters.

    PubMed

    Lai, Charles P; Kim, Edward Y; Badr, Christian E; Weissleder, Ralph; Mempel, Thorsten R; Tannous, Bakhos A; Breakefield, Xandra O

    2015-01-01

    Accurate spatiotemporal assessment of extracellular vesicle (EV) delivery and cargo RNA translation requires specific and robust live-cell imaging technologies. Here we engineer optical reporters to label multiple EV populations for visualization and tracking of tumour EV release, uptake and exchange between cell populations both in culture and in vivo. Enhanced green fluorescence protein (EGFP) and tandem dimer Tomato (tdTomato) were fused at NH2-termini with a palmitoylation signal (PalmGFP, PalmtdTomato) for EV membrane labelling. To monitor EV-RNA cargo, transcripts encoding PalmtdTomato were tagged with MS2 RNA binding sequences and detected by co-expression of bacteriophage MS2 coat protein fused with EGFP. By multiplexing fluorescent and bioluminescent EV membrane reporters, we reveal the rapid dynamics of both EV uptake and translation of EV-delivered cargo mRNAs in cancer cells that occurred within 1-hour post-horizontal transfer between cells. These studies confirm that EV-mediated communication is dynamic and multidirectional between cells with delivery of functional mRNA. PMID:25967391

  16. Giant magnetoresistive sensor array for sensitive and specific multiplexed food allergen detection.

    PubMed

    Ng, Elaine; Nadeau, Kari C; Wang, Shan X

    2016-06-15

    Current common allergen detection methods, including enzyme-linked immunosorbent assays (ELISAs) and dip-stick methods, do not provide adequate levels of sensitivity and specificity for at-risk allergic patients. A method for performing highly sensitive and specific detection of multiple food allergens is thus imperative as food allergies are becoming increasingly recognized as a major healthcare concern, affecting an estimated 4% of the total population. We demonstrate first instance of sensitive and specific multiplexed detection of major peanut allergens Ara h 1 and Ara h 2, and wheat allergen Gliadin using giant magnetoresistive (GMR) sensor arrays. Commercialized ELISA kits for Ara h 1 and Ara h 2 report limits of detection (LODs) at 31.5 ng/mL and 0.2 ng/mL, respectively. In addition, the 96-well-based ELISA developed in-house for Gliadin was found to have a LOD of 40 ng/mL. Our multiplexed GMR-based assay demonstrates the ability to perform all three assays on the same chip specifically and with sensitivities at LODs about an order of magnitude lower than those of 96-well-based ELISAs. LODs of GMR-based assays developed for Ara h 1, Ara h 2, and Gliadin were 7.0 ng/mL, 0.2 ng/mL, and 1.5 ng/mL, respectively, with little to no cross-reactivity. These LODs are clinically important as some patients could react strongly against such low allergen levels. Given the limitations of current industrial detection technology, multiplexed GMR-based assays provide a method for highly sensitive and specific simultaneous detection of any combination of food-product allergens, thus protecting allergic patients from life-threatening events, including anaphylaxis, by unintentional consumption.

  17. Giant magnetoresistive sensor array for sensitive and specific multiplexed food allergen detection.

    PubMed

    Ng, Elaine; Nadeau, Kari C; Wang, Shan X

    2016-06-15

    Current common allergen detection methods, including enzyme-linked immunosorbent assays (ELISAs) and dip-stick methods, do not provide adequate levels of sensitivity and specificity for at-risk allergic patients. A method for performing highly sensitive and specific detection of multiple food allergens is thus imperative as food allergies are becoming increasingly recognized as a major healthcare concern, affecting an estimated 4% of the total population. We demonstrate first instance of sensitive and specific multiplexed detection of major peanut allergens Ara h 1 and Ara h 2, and wheat allergen Gliadin using giant magnetoresistive (GMR) sensor arrays. Commercialized ELISA kits for Ara h 1 and Ara h 2 report limits of detection (LODs) at 31.5 ng/mL and 0.2 ng/mL, respectively. In addition, the 96-well-based ELISA developed in-house for Gliadin was found to have a LOD of 40 ng/mL. Our multiplexed GMR-based assay demonstrates the ability to perform all three assays on the same chip specifically and with sensitivities at LODs about an order of magnitude lower than those of 96-well-based ELISAs. LODs of GMR-based assays developed for Ara h 1, Ara h 2, and Gliadin were 7.0 ng/mL, 0.2 ng/mL, and 1.5 ng/mL, respectively, with little to no cross-reactivity. These LODs are clinically important as some patients could react strongly against such low allergen levels. Given the limitations of current industrial detection technology, multiplexed GMR-based assays provide a method for highly sensitive and specific simultaneous detection of any combination of food-product allergens, thus protecting allergic patients from life-threatening events, including anaphylaxis, by unintentional consumption. PMID:26859787

  18. mRNA profiling for body fluid identification by multiplex quantitative RT-PCR.

    PubMed

    Juusola, Jane; Ballantyne, Jack

    2007-11-01

    An alternative approach to conventional protein-based body fluid identification is gene expression profiling analysis. In the present work, we report the development of sensitive and robust multiplex quantitative reverse transcriptase-PCR assays for the identification of blood, saliva, semen, and menstrual blood. Each body fluid assay comprises a triplex system that detects transcripts from two body fluid-specific genes and a housekeeping gene GAPDH. The body fluid-specific genes include erythroid delta-aminolevulinate synthase (ALAS2) and beta-spectrin (SPTB) for blood, statherin (STATH) and histatin 3 (HTN3) for saliva, protamine 1 (PRM1) and protamine 2 (PRM2) for semen, and matrix metalloproteinase 7 (MMP7) and matrix metalloproteinase 10 (MMP10) for menstrual blood. Normalization of both body fluid-specific genes to the housekeeping gene by means of appropriate cycle threshold metrics ensures the high specificity of each assay for its cognate body fluid.

  19. Capillary electrophoresis of a multiplex reverse transcription-polymerase chain reaction to target messenger RNA markers for body fluid identification.

    PubMed

    Haas, Cordula; Hanson, Erin; Ballantyne, Jack

    2012-01-01

    The analysis of cell-specific mRNA expression is a promising new method for the identification of body fluids. A number of mRNA markers have been identified for the forensically most relevant body fluids: blood, saliva, semen, vaginal secretions, and menstrual blood. Apart from a significant improvement in specificity compared to conventional protein-based methods, other important advantages of body fluid identification by mRNA profiling include the possibility of simultaneously isolating RNA and DNA from the same piece of stain and the ability to multiplex numerous RNA markers for the identification of one or several body fluids. RNA profiling can be incorporated into current DNA analysis pipelines.

  20. 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

  1. 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

  2. A multiplex readout method for position sensitive boron coated straw neutron detector

    NASA Astrophysics Data System (ADS)

    Yu, Hao; Gong, Hui; Li, Jianmin; Wang, Yongqiang; Wang, Xuewu; Li, Yuanjing; Kang, Kejun

    2015-10-01

    A 1 m×1 m boron coated straw neutron detector is expected to be used to build the small-angle neutron scattering (SANS) instrument of the Compact Pulsed Hadron Source (CPHS) in Tsinghua University. A multiplex readout method based on summing circuits in columns and rows is studied for this large area position sensitive detector. In this method, the outputs of charge sensitive preamplifiers are combined by columns and rows at two ends of the detector, and then the shaped signals are sampled by flash ADCs. With the position reconstructed algorithm implemented in FPGA which analyzes the charge division and column and row number of signals, the 3-D position information of neutron events can be obtained. The position resolution and counting rate performance of this method are analyzed, and the comparison to the delay-line readout method is also given. With the multiplex readout method, the scale of readout electronics can be greatly reduced and a good position resolution can be reached. A readout electronics system for a detector module which consists 4 × 10 straw tubes is designed based on this method, and the test with neutron beam shows an average 3-D spatial resolution of 4 × 4 × 6.8mm3.

  3. Multiplex Detection of Rare Mutations by Picoliter Droplet Based Digital PCR: Sensitivity and Specificity Considerations

    PubMed Central

    Zonta, Eleonora; Garlan, Fanny; Pécuchet, Nicolas; Perez-Toralla, Karla; Caen, Ouriel; Milbury, Coren; Didelot, Audrey; Fabre, Elizabeth; Blons, Hélène; Laurent-Puig, Pierre; Taly, Valérie

    2016-01-01

    In cancer research, the accuracy of the technology used for biomarkers detection is remarkably important. In this context, digital PCR represents a highly sensitive and reproducible method that could serve as an appropriate tool for tumor mutational status analysis. In particular, droplet-based digital PCR approaches have been developed for detection of tumor-specific mutated alleles within plasmatic circulating DNA. Such an approach calls for the development and validation of a very significant quantity of assays, which can be extremely costly and time consuming. Herein, we evaluated assays for the detection and quantification of various mutations occurring in three genes often misregulated in cancers: the epidermal growth factor receptor (EGFR), the v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) and the Tumoral Protein p53 (TP53) genes. In particular, commercial competitive allele-specific TaqMan® PCR (castPCR™) technology, as well as TaqMan® and ZEN™ assays, have been evaluated for EGFR p.L858R, p.T790M, p.L861Q point mutations and in-frame deletions Del19. Specificity and sensitivity have been determined on cell lines DNA, plasmatic circulating DNA of lung cancer patients or Horizon Diagnostics Reference Standards. To show the multiplexing capabilities of this technology, several multiplex panels for EGFR (several three- and four-plexes) have been developed, offering new "ready-to-use" tests for lung cancer patients. PMID:27416070

  4. Plasmonic substrates for multiplexed protein microarrays with femtomolar sensitivity and broad dynamic range

    PubMed Central

    Tabakman, Scott M.; Lau, Lana; Robinson, Joshua T.; Price, Jordan; Sherlock, Sarah P.; Wang, Hailiang; Zhang, Bo; Chen, Zhuo; Tangsombatvisit, Stephanie; Jarrell, Justin A.; Utz, Paul J.; Dai, Hongjie

    2012-01-01

    Protein chips are widely used for high-throughput proteomic analysis, but to date, the low sensitivity and narrow dynamic range have limited their capabilities in diagnostics and proteomics. Here we present protein microarrays on a novel nanostructured, plasmonic gold film with near-infrared fluorescence enhancement of up to 100-fold, extending the dynamic range of protein detection by three orders of magnitude towards the fM regime. We employ plasmonic protein microarrays for the early detection of a cancer biomarker, carcinoembryonic antigen, in the sera of mice bearing a xenograft tumour model. Further, we demonstrate a multiplexed autoantigen array for human autoantibodies implicated in a range of autoimmune diseases with superior signal-to-noise ratios and broader dynamic range compared with commercial nitrocellulose and glass substrates. The high sensitivity, broad dynamic range and easy adaptability of plasmonic protein chips presents new opportunities in proteomic research and diagnostics applications. PMID:21915108

  5. Large area CMOS bio-pixel array for compact high sensitive multiplex biosensing.

    PubMed

    Sandeau, Laure; Vuillaume, Cassandre; Contié, Sylvain; Grinenval, Eva; Belloni, Federico; Rigneault, Hervé; Owens, Roisin M; Fournet, Margaret Brennan

    2015-02-01

    A novel CMOS bio-pixel array which integrates assay substrate and assay readout is demonstrated for multiplex and multireplicate detection of a triplicate of cytokines with single digit pg ml(-1) sensitivities. Uniquely designed large area bio-pixels enable individual assays to be dedicated to and addressed by single pixels. A capability to simultaneously measure a large number of targets is provided by the 128 available pixels. Chemiluminescent assays are carried out directly on the pixel surface which also detects the emitted chemiluminescent photons, facilitating a highly compact sensor and reader format. The high sensitivity of the bio-pixel array is enabled by the high refractive index of silicon based pixels. This in turn generates a strong supercritical angle luminescence response significantly increasing the efficiency of the photon collection over conventional farfield modalities. PMID:25490928

  6. Highly Multiplexed RNA Aptamer Selection using a Microplate-based Microcolumn Device

    PubMed Central

    Reinholt, Sarah J.; Ozer, Abdullah; Lis, John T.; Craighead, Harold G.

    2016-01-01

    We describe a multiplexed RNA aptamer selection to 19 different targets simultaneously using a microcolumn-based device, MEDUSA (Microplate-based Enrichment Device Used for the Selection of Aptamers), as well as a modified selection process, that significantly reduce the time and reagents needed for selections. We exploited MEDUSA’s reconfigurable design between parallel and serially-connected microcolumns to enable the use of just 2 aliquots of starting library, and its 96-well microplate compatibility to enable the continued use of high-throughput techniques in downstream processes. Our modified selection protocol allowed us to perform the equivalent of a 10-cycle selection in the time it takes for 4 traditional selection cycles. Several aptamers were discovered with nanomolar dissociation constants. Furthermore, aptamers were identified that not only bound with high affinity, but also acted as inhibitors to significantly reduce the activity of their target protein, mouse decapping exoribonuclease (DXO). The aptamers resisted DXO’s exoribonuclease activity, and in studies monitoring DXO’s degradation of a 30-nucleotide substrate, less than 1 μM of aptamer demonstrated significant inhibition of DXO activity. This aptamer selection method using MEDUSA helps to overcome some of the major challenges with traditional aptamer selections, and provides a platform for high-throughput selections that lends itself to process automation. PMID:27432610

  7. Enhanced sensitivity RNA gel loading buffer that enables efficient RNA separation on native gels.

    PubMed

    Gregg, Keqin; Zhou, Wenli; Ji, Wan; Davis, Sara

    2004-02-01

    RNA gel analysis is essential for quality assessment of RNA preparations for subsequent analysis such as microarrays and real-time PCRs. The routinely used standard electrophoresis of RNA through formaldehyde-containing agarose gels is not only labor-intensive and time-consuming, but also involves sizeable quantities of hazardous materials. Above all, it is not sensitive, requiring more than 1 microgram of RNA for the assay. Current gene expression profiling with microarrays and real-time PCR often involves limiting amounts of RNA. It is therefore important to have a more sensitive way to analyze RNA. Here we report an improved ethidium bromide-based RNA gel analysis system with our Superload buffer that increases sensitivity to 12.5 ng of total RNA and allows RNA analysis on a regular native Tris-acetate EDTA (TAE) agarose gel.

  8. Simple, Sensitive and Accurate Multiplex Detection of Clinically Important Melanoma DNA Mutations in Circulating Tumour DNA with SERS Nanotags

    PubMed Central

    Wee, Eugene J.H.; Wang, Yuling; Tsao, Simon Chang-Hao; Trau, Matt

    2016-01-01

    Sensitive and accurate identification of specific DNA mutations can influence clinical decisions. However accurate diagnosis from limiting samples such as circulating tumour DNA (ctDNA) is challenging. Current approaches based on fluorescence such as quantitative PCR (qPCR) and more recently, droplet digital PCR (ddPCR) have limitations in multiplex detection, sensitivity and the need for expensive specialized equipment. Herein we describe an assay capitalizing on the multiplexing and sensitivity benefits of surface-enhanced Raman spectroscopy (SERS) with the simplicity of standard PCR to address the limitations of current approaches. This proof-of-concept method could reproducibly detect as few as 0.1% (10 copies, CV < 9%) of target sequences thus demonstrating the high sensitivity of the method. The method was then applied to specifically detect three important melanoma mutations in multiplex. Finally, the PCR/SERS assay was used to genotype cell lines and ctDNA from serum samples where results subsequently validated with ddPCR. With ddPCR-like sensitivity and accuracy yet at the convenience of standard PCR, we believe this multiplex PCR/SERS method could find wide applications in both diagnostics and research. PMID:27446486

  9. Analysis of sensitivity and rapid hybridization of a multiplexed Microbial Detection Microarray

    SciTech Connect

    Thissen, James B.; McLoughlin, Kevin; Gardner, Shea; Gu, Pauline; Mabery, Shalini; Slezak, Tom; Jaing, Crystal

    2014-06-01

    Microarrays have proven to be useful in rapid detection of many viruses and bacteria. Pathogen detection microarrays have been used to diagnose viral and bacterial infections in clinical samples and to evaluate the safety of biological drug materials. A multiplexed version of the Lawrence Livermore Microbial Detection Array (LLMDA) was developed and evaluated with minimum detectable concentrations for pure unamplified DNA viruses, along with mixtures of viral and bacterial DNA subjected to different whole genome amplification protocols. In addition the performance of the array was tested when hybridization time was reduced from 17 h to 1 h. The LLMDA was able to detect unamplified vaccinia virus DNA at a concentration of 14 fM, or 100,000 genome copies in 12 μL of sample. With amplification, positive identification was made with only 100 genome copies of input material. When tested against human stool samples from patients with acute gastroenteritis, the microarray detected common gastroenteritis viral and bacterial infections such as rotavirus and E. coli. Accurate detection was found but with a 4-fold drop in sensitivity for a 1 h compared to a 17 h hybridization. The array detected 2 ng (equivalent concentration of 15.6 fM) of labeled DNA from a virus with 1 h hybridization without any amplification, and was able to identify the components of a mixture of viruses and bacteria at species and in some cases strain level resolution. Sensitivity improved by three orders of magnitude with random whole genome amplification prior to hybridization; for instance, the array detected a DNA virus with only 20 fg or 100 genome copies as input. This multiplexed microarray is an efficient tool to analyze clinical and environmental samples for the presence of multiple viral and bacterial pathogens rapidly.

  10. Analysis of sensitivity and rapid hybridization of a multiplexed Microbial Detection Microarray

    DOE PAGES

    Thissen, James B.; McLoughlin, Kevin; Gardner, Shea; Gu, Pauline; Mabery, Shalini; Slezak, Tom; Jaing, Crystal

    2014-06-01

    Microarrays have proven to be useful in rapid detection of many viruses and bacteria. Pathogen detection microarrays have been used to diagnose viral and bacterial infections in clinical samples and to evaluate the safety of biological drug materials. A multiplexed version of the Lawrence Livermore Microbial Detection Array (LLMDA) was developed and evaluated with minimum detectable concentrations for pure unamplified DNA viruses, along with mixtures of viral and bacterial DNA subjected to different whole genome amplification protocols. In addition the performance of the array was tested when hybridization time was reduced from 17 h to 1 h. The LLMDA wasmore » able to detect unamplified vaccinia virus DNA at a concentration of 14 fM, or 100,000 genome copies in 12 μL of sample. With amplification, positive identification was made with only 100 genome copies of input material. When tested against human stool samples from patients with acute gastroenteritis, the microarray detected common gastroenteritis viral and bacterial infections such as rotavirus and E. coli. Accurate detection was found but with a 4-fold drop in sensitivity for a 1 h compared to a 17 h hybridization. The array detected 2 ng (equivalent concentration of 15.6 fM) of labeled DNA from a virus with 1 h hybridization without any amplification, and was able to identify the components of a mixture of viruses and bacteria at species and in some cases strain level resolution. Sensitivity improved by three orders of magnitude with random whole genome amplification prior to hybridization; for instance, the array detected a DNA virus with only 20 fg or 100 genome copies as input. This multiplexed microarray is an efficient tool to analyze clinical and environmental samples for the presence of multiple viral and bacterial pathogens rapidly.« less

  11. Rapid and Sensitive PCR-Dipstick DNA Chromatography for Multiplex Analysis of the Oral Microbiota

    PubMed Central

    Niwa, Kousuke; Kawase, Mitsuo; Tanner, Anne C. R.; Takahashi, Nobuhiro

    2014-01-01

    A complex of species has been associated with dental caries under the ecological hypothesis. This study aimed to develop a rapid, sensitive PCR-dipstick DNA chromatography assay that could be read by eye for multiplex and semiquantitative analysis of plaque bacteria. Parallel oligonucleotides were immobilized on a dipstick strip for multiplex analysis of target DNA sequences of the caries-associated bacteria, Streptococcus mutans, Streptococcus sobrinus, Scardovia wiggsiae, Actinomyces species, and Veillonella parvula. Streptavidin-coated blue-colored latex microspheres were to generate signal. Target DNA amplicons with an oligonucleotide-tagged terminus and a biotinylated terminus were coupled with latex beads through a streptavidin-biotin interaction and then hybridized with complementary oligonucleotides on the strip. The accumulation of captured latex beads on the test and control lines produced blue bands, enabling visual detection with the naked eye. The PCR-dipstick DNA chromatography detected quantities as low as 100 pg of DNA amplicons and demonstrated 10- to 1000-fold higher sensitivity than PCR-agarose gel electrophoresis, depending on the target bacterial species. Semiquantification of bacteria was performed by obtaining a series of chromatograms using serial 10-fold dilution of PCR-amplified DNA extracted from dental plaque samples. The assay time was less than 3 h. The semiquantification procedure revealed the relative amounts of each test species in dental plaque samples, indicating that this disposable device has great potential in analysis of microbial composition in the oral cavity and intestinal tract, as well as in point-of-care diagnosis of microbiota-associated diseases. PMID:25485279

  12. Engineered nanoconstructs for the multiplexed and sensitive detection of high-risk pathogens

    NASA Astrophysics Data System (ADS)

    Seo, Youngmin; Kim, Ji-Eun; Jeong, Yoon; Lee, Kwan Hong; Hwang, Jangsun; Hong, Jongwook; Park, Hansoo; Choi, Jonghoon

    2016-01-01

    Many countries categorize the causative agents of severe infectious diseases as high-risk pathogens. Given their extreme infectivity and potential to be used as biological weapons, a rapid and sensitive method for detection of high-risk pathogens (e.g., Bacillus anthracis, Francisella tularensis, Yersinia pestis, and Vaccinia virus) is highly desirable. Here, we report the construction of a novel detection platform comprising two units: (1) magnetic beads separately conjugated with multiple capturing antibodies against four different high-risk pathogens for simple and rapid isolation, and (2) genetically engineered apoferritin nanoparticles conjugated with multiple quantum dots and detection antibodies against four different high-risk pathogens for signal amplification. For each high-risk pathogen, we demonstrated at least 10-fold increase in sensitivity compared to traditional lateral flow devices that utilize enzyme-based detection methods. Multiplexed detection of high-risk pathogens in a sample was also successful by using the nanoconstructs harboring the dye molecules with fluorescence at different wavelengths. We ultimately envision the use of this novel nanoprobe detection platform in future applications that require highly sensitive on-site detection of high-risk pathogens.

  13. Genome-wide mRNA profiling and multiplex quantitative RT-PCR for forensic body fluid identification.

    PubMed

    Park, Seong-Min; Park, Seong-Yeon; Kim, Jeong-Hwan; Kang, Tae-Wook; Park, Jong-Lyul; Woo, Kwang-Man; Kim, Jong-Sik; Lee, Han-Chul; Kim, Seon-Young; Lee, Seung-Hwan

    2013-01-01

    In forensic science, identifying a tissue where a forensic specimen was originated is one of the principal challenges. Messenger RNA (mRNA) profile clearly reveals tissue-specific gene expression patterns that many attempts have been made to use RNA for forensic tissue identification. To systematically investigate the body-fluid-specific expression of mRNAs and find novel mRNA markers for forensic body fluid identification, we performed DNA microarray experiment with 24 Korean body fluid samples. Shannon entropy and Q-values were calculated for each gene, and 137 body-fluid-specific candidate genes were selected. By applying more stringent criteria, we further selected 28 candidate genes and validated them by RT-PCR and qRT-PCR. As a result, we suggest a novel combination of four body-fluid-specific mRNA makers: PPBP for blood, FDCSP for saliva, MSMB for semen and MSLN for vaginal secretion. Multiplex qRT-PCR assay was designed using the four mRNA markers and DNA/RNA co-extraction method was tested for forensic use. This study will provide a thorough examination of body-fluid-specifically expressed mRNAs, which will enlarge the possibility of practical use of RNA for forensic purpose.

  14. High-speed polarization sensitive optical frequency domain imaging with frequency multiplexing.

    PubMed

    Oh, W Y; Yun, S H; Vakoc, B J; Shishkov, M; Desjardins, A E; Park, B H; de Boer, J F; Tearney, G J; Bouma, B E

    2008-01-21

    Polarization sensitive optical coherence tomography (PS-OCT) provides a cross-sectional image of birefringence in biological samples that is complementary in many applications to the standard reflectance-based image. Recent ex vivo studies have demonstrated that birefringence mapping enables the characterization of collagen and smooth muscle concentration and distribution in vascular tissues. Instruments capable of applying these measurements percutaneously in vivo may provide new insights into coronary atherosclerosis and acute myocardial infarction. We have developed a polarization sensitive optical frequency domain imaging (PS-OFDI) system that enables high-speed intravascular birefringence imaging through a fiber-optic catheter. The novel design of this system utilizes frequency multiplexing to simultaneously measure reflectance of two incident polarization states, overcoming concerns regarding temporal variations of the catheter fiber birefringence and spatial variations in the birefringence of the sample. We demonstrate circular cross-sectional birefringence imaging of a human coronary artery ex vivo through a flexible fiber-optic catheter with an A-line rate of 62 kHz and a ranging depth of 6.2 mm.

  15. Examining the Interactions of the Splicing Factor MBNL1 with Target RNA Sequences via a Label-Free, Multiplex Method

    PubMed Central

    Yadav, Amrita R.; Mace, Charles R.; Miller, Benjamin L.

    2014-01-01

    The near-ubiquity of the involvement of RNA in crucial biological processes is accepted. It is important, therefore, to study and understand the biophysical principles that regulate the function of RNA and its interactions with other molecules (e.g., proteins and antibiotics). Methods enabling the high-throughput determination of RNA–protein binding kinetics and thermodynamics would greatly accelerate understanding of these interactions. To that end, we describe the development of a real-time biomolecular interaction analysis platform based on arrayed imaging reflectometry (AIR) for multiplex analysis of RNA–protein interactions. We demonstrate the use of aqueous AIR by measuring the binding kinetics between muscleblind-like 1 (MBNL1), a splicing regulator protein that plays a pivotal role in the Myotonic Dystrophies and Huntington's Disease, and several of its RNA targets simultaneously on a microarrayed chip. Using this approach, we observe that the kinetics of MBNL1 binding isolated CUG and repeat CUG RNA sequences (as models for “normal” and “pathogenic” RNA, respectively) are different even though their steady state binding constants are similar. The ability to compare binding kinetics between RNA sequences rapidly and easily may provide insight into the molecular basis of MBNL1-RNA binding, and more generally suggests that AIR can be a powerful tool to enable the label-free, real-time analysis of biomolecular interactions in a high throughput format. PMID:24377303

  16. In vivo mutagenesis of miRNA gene families using a scalable multiplexed CRISPR/Cas9 nuclease system

    PubMed Central

    Narayanan, Anand; Hill-Teran, Guillermina; Moro, Albertomaria; Ristori, Emma; Kasper, Dionna M.; A. Roden, Christine; Lu, Jun; Nicoli, Stefania

    2016-01-01

    A large number of microRNAs (miRNAs) are grouped into families derived from the same phylogenetic ancestors. miRNAs within a family often share the same physiological functions despite differences in their primary sequences, secondary structures, or chromosomal locations. Consequently, the generation of animal models to analyze the activity of miRNA families is extremely challenging. Using zebrafish as a model system, we successfully provide experimental evidence that a large number of miRNAs can be simultaneously mutated to abrogate the activity of an entire miRNA family. We show that injection of the Cas9 nuclease and two, four, ten, and up to twenty-four multiplexed single guide RNAs (sgRNAs) can induce mutations in 90% of the miRNA genomic sequences analyzed. We performed a survey of these 45 mutations in 10 miRNA genes, analyzing the impact of our mutagenesis strategy on the processing of each miRNA both computationally and in vivo. Our results offer an effective approach to mutate and study the activity of miRNA families and pave the way for further analysis on the function of complex miRNA families in higher multicellular organisms. PMID:27572667

  17. siRNA Delivery by Stimuli-Sensitive Nanocarriers

    PubMed Central

    Salzano, Giuseppina; Costa, Daniel F.; Torchilin, Vladimir P.

    2016-01-01

    Since its discovery in late 1990s, small interfering RNA (siRNA) has become a significant biopharmaceutical research tool and a powerful option for the treatment of different human diseases based on altered gene-expression. Despite promising data from many pre-clinical studies, concrete hurdles still need to be overcome to bring therapeutic siRNAs in clinic. The design of stimuli-sensitive nanopreparations for gene therapy is a lively area of the current research. Compared to conventional systems for siRNA delivery, this type of platform can respond to local stimuli that are characteristics of the pathological area of interest, allowing the release of nucleic acids at the desired site. Acidic pH, abnormal levels of enzymes, altered redox potential and magnetic field are examples of stimuli exploited in the design of stimuli-sensitive nanoparticles. In this review, we discuss on recent stimuli-sensitive strategies for siRNA delivery and we highlight on the potential of combining multiple stimuli-sensitive strategies in the same nano-platform for a better therapeutic outcome. PMID:26486143

  18. Evaluation of Multiplexed 16S rRNA Microbial Population Surveys Using Illumina MiSeq Platform (Seventh Annual Sequencing, Finishing, Analysis in the Future (SFAF) Meeting 2012)

    ScienceCinema

    Tremblay, Julien [DOE JGI

    2016-07-12

    Julien Tremblay from DOE JGI presents "Evaluation of Multiplexed 16S rRNA Microbial Population Surveys Using Illumina MiSeq Platorm" at the 7th Annual Sequencing, Finishing, Analysis in the Future (SFAF) Meeting held in June, 2012 in Santa Fe, NM.

  19. Evaluation of Multiplexed 16S rRNA Microbial Population Surveys Using Illumina MiSeq Platform (Seventh Annual Sequencing, Finishing, Analysis in the Future (SFAF) Meeting 2012)

    SciTech Connect

    Tremblay, Julien

    2012-06-01

    Julien Tremblay from DOE JGI presents "Evaluation of Multiplexed 16S rRNA Microbial Population Surveys Using Illumina MiSeq Platorm" at the 7th Annual Sequencing, Finishing, Analysis in the Future (SFAF) Meeting held in June, 2012 in Santa Fe, NM.

  20. siRNA Delivery by Stimuli-Sensitive Nanocarriers.

    PubMed

    Salzano, Giuseppina; Costa, Daniel F; Torchilin, Vladimir P

    2015-01-01

    Since its discovery in the late 1990, small interfering RNA (siRNA) have quickly crept into the biopharmaceutical research as a new and powerful tool for the treatment of different human diseases based on altered gene-expression. Despite promising data from many pre-clinical studies, concrete hurdles still need to be overcome to bring therapeutic siRNAs in clinic. The design of stimuli-sensitive nanopreparations for gene therapy is a lively area of the current research. Compared to conventional systems for siRNA delivery, this type of platform can respond to local stimuli that are characteristics of the pathological area of interest, allowing the release of nucleic acids at the desired site. Acidic pH, de-regulated levels of enzymes, altered redox potential and magnetic field are examples of stimuli exploit to design stimuli-sensitive nanoparticles. In this review, we discuss on recent stimulisensitive strategies for siRNA delivery and we highlight on the potential of combining multiple stimuli-sensitive strategies in the same nano-platform for a better therapeutic outcome. PMID:26486143

  1. Automated Methods for Multiplexed Pathogen Detection

    SciTech Connect

    Straub, Tim M.; Dockendorff, Brian P.; Quinonez-Diaz, Maria D.; Valdez, Catherine O.; Shutthanandan, Janani I.; Tarasevich, Barbara J.; Grate, Jay W.; Bruckner-Lea, Cindy J.

    2005-09-01

    Detection of pathogenic microorganisms in environmental samples is a difficult process. Concentration of the organisms of interest also co-concentrates inhibitors of many end-point detection methods, notably, nucleic acid methods. In addition, sensitive, highly multiplexed pathogen detection continues to be problematic. The primary function of the BEADS (Biodetection Enabling Analyte Delivery System) platform is the automated concentration and purification of target analytes from interfering substances, often present in these samples, via a renewable surface column. In one version of BEADS, automated immunomagnetic separation (IMS) is used to separate cells from their samples. Captured cells are transferred to a flow-through thermal cycler where PCR, using labeled primers, is performed. PCR products are then detected by hybridization to a DNA suspension array. In another version of BEADS, cell lysis is performed, and community RNA is purified and directly labeled. Multiplexed detection is accomplished by direct hybridization of the RNA to a planar microarray. The integrated IMS/PCR version of BEADS can successfully purify and amplify 10 E. coli O157:H7 cells from river water samples. Multiplexed PCR assays for the simultaneous detection of E. coli O157:H7, Salmonella, and Shigella on bead suspension arrays was demonstrated for the detection of as few as 100 cells for each organism. Results for the RNA version of BEADS are also showing promising results. Automation yields highly purified RNA, suitable for multiplexed detection on microarrays, with microarray detection specificity equivalent to PCR. Both versions of the BEADS platform show great promise for automated pathogen detection from environmental samples. Highly multiplexed pathogen detection using PCR continues to be problematic, but may be required for trace detection in large volume samples. The RNA approach solves the issues of highly multiplexed PCR and provides ''live vs. dead'' capabilities. However

  2. Analytical Sensitivity Comparison between Singleplex Real-Time PCR and a Multiplex PCR Platform for Detecting Respiratory Viruses

    PubMed Central

    Parker, Jayme; Fowler, Nisha; Walmsley, Mary Louise; Schmidt, Terri; Scharrer, Jason; Kowaleski, James; Grimes, Teresa; Hoyos, Shanann; Chen, Jack

    2015-01-01

    Multiplex PCR methods are attractive to clinical laboratories wanting to broaden their detection of respiratory viral pathogens in clinical specimens. However, multiplexed assays must be well optimized to retain or improve upon the analytic sensitivity of their singleplex counterparts. In this experiment, the lower limit of detection (LOD) of singleplex real-time PCR assays targeting respiratory viruses is compared to an equivalent panel on a multiplex PCR platform, the GenMark eSensor RVP. LODs were measured for each singleplex real-time PCR assay and expressed as the lowest copy number detected 95–100% of the time, depending on the assay. The GenMark eSensor RVP LODs were obtained by converting the TCID50/mL concentrations reported in the package insert to copies/μL using qPCR. Analytical sensitivity between the two methods varied from 1.2–1280.8 copies/μL (0.08–3.11 log differences) for all 12 assays compared. Assays targeting influenza A/H3N2, influenza A/H1N1pdm09, influenza B, and human parainfluenza 1 and 2 were most comparable (1.2–8.4 copies/μL, <1 log difference). Largest differences in LOD were demonstrated for assays targeting adenovirus group E, respiratory syncytial virus subtype A, and a generic assay for all influenza A viruses regardless of subtype (319.4–1280.8 copies/μL, 2.50–3.11 log difference). The multiplex PCR platform, the GenMark eSensor RVP, demonstrated improved analytical sensitivity for detecting influenza A/H3 viruses, influenza B virus, human parainfluenza virus 2, and human rhinovirus (1.6–94.8 copies/μL, 0.20–1.98 logs). Broader detection of influenza A/H3 viruses was demonstrated by the GenMark eSensor RVP. The relationship between TCID50/mL concentrations and the corresponding copy number related to various ATCC cultures is also reported. PMID:26569120

  3. Multiplex bioimaging of piRNA molecular pathway-regulated theragnostic effects in a single breast cancer cell using a piRNA molecular beacon.

    PubMed

    Lee, Youn Jung; Moon, Sung Ung; Park, Min Geun; Jung, Woon Yong; Park, Yong Keun; Song, Sung Kyu; Ryu, Je Gyu; Lee, Yong Seung; Heo, Hye Jung; Gu, Ha Na; Cho, Su Jeong; Ali, Bahy A; Al-Khedhairy, Abdulaziz A; Lee, Ilkyun; Kim, Soonhag

    2016-09-01

    Recently, PIWI-interacting small non-coding RNAs (piRNAs) have emerged as novel cancer biomarkers candidate because of their high expression level in various cancer types and role in the control of tumor suppressor genes. In this study, a novel breast cancer theragnostics probe based on a single system targeting the piRNA-36026 (piR-36026) molecular pathway was developed using a piR-36026 molecular beacon (MB). The piR-36026 MB successfully visualized endogenous piR-36026 biogenesis, which is highly expressed in MCF7 cells (a human breast cancer cell line), and simultaneously inhibited piR-36026-mediated cancer progression in vitro and in vivo. We discovered two tumor suppressor proteins, SERPINA1 and LRAT, that were directly regulated as endogenous piR-36026 target genes in MCF7 cells. Furthermore, multiplex bioimaging of a single MCF7 cell following treatment with piR-36026 MB clearly visualized the direct molecular interaction of piRNA-36026 with SERPINA1 or LRAT and subsequent molecular therapeutic responses including caspase-3 and PI in the nucleus. PMID:27289065

  4. Multiplex bioimaging of piRNA molecular pathway-regulated theragnostic effects in a single breast cancer cell using a piRNA molecular beacon.

    PubMed

    Lee, Youn Jung; Moon, Sung Ung; Park, Min Geun; Jung, Woon Yong; Park, Yong Keun; Song, Sung Kyu; Ryu, Je Gyu; Lee, Yong Seung; Heo, Hye Jung; Gu, Ha Na; Cho, Su Jeong; Ali, Bahy A; Al-Khedhairy, Abdulaziz A; Lee, Ilkyun; Kim, Soonhag

    2016-09-01

    Recently, PIWI-interacting small non-coding RNAs (piRNAs) have emerged as novel cancer biomarkers candidate because of their high expression level in various cancer types and role in the control of tumor suppressor genes. In this study, a novel breast cancer theragnostics probe based on a single system targeting the piRNA-36026 (piR-36026) molecular pathway was developed using a piR-36026 molecular beacon (MB). The piR-36026 MB successfully visualized endogenous piR-36026 biogenesis, which is highly expressed in MCF7 cells (a human breast cancer cell line), and simultaneously inhibited piR-36026-mediated cancer progression in vitro and in vivo. We discovered two tumor suppressor proteins, SERPINA1 and LRAT, that were directly regulated as endogenous piR-36026 target genes in MCF7 cells. Furthermore, multiplex bioimaging of a single MCF7 cell following treatment with piR-36026 MB clearly visualized the direct molecular interaction of piRNA-36026 with SERPINA1 or LRAT and subsequent molecular therapeutic responses including caspase-3 and PI in the nucleus.

  5. Multiplex Real-Time qPCR Assay for Simultaneous and Sensitive Detection of Phytoplasmas in Sesame Plants and Insect Vectors

    PubMed Central

    Ikten, Cengiz; Ustun, Rustem; Catal, Mursel; Yol, Engin; Uzun, Bulent

    2016-01-01

    Phyllody, a destructive and economically important disease worldwide caused by phytoplasma infections, is characterized by the abnormal development of floral structures into stunted leafy parts and contributes to serious losses in crop plants, including sesame (Sesamum indicum L.). Accurate identification, differentiation, and quantification of phyllody-causing phytoplasmas are essential for effective management of this plant disease and for selection of resistant sesame varieties. In this study, a diagnostic multiplex qPCR assay was developed using TaqMan® chemistry based on detection of the 16S ribosomal RNA gene of phytoplasmas and the 18S ribosomal gene of sesame. Phytoplasma and sesame specific primers and probes labeled with different fluorescent dyes were used for simultaneous amplification of 16SrII and 16SrIX phytoplasmas in a single tube. The multiplex real-time qPCR assay allowed accurate detection, differentiation, and quantification of 16SrII and 16SrIX groups in 109 sesame plant and 92 insect vector samples tested. The assay was found to have a detection sensitivity of 1.8 x 102 and 1.6 x 102 DNA copies for absolute quantification of 16SrII and 16SrIX group phytoplasmas, respectively. Relative quantification was effective and reliable for determination of phyllody phytoplasma DNA amounts normalized to sesame DNA in infected plant tissues. The development of this qPCR assay provides a method for the rapid measurement of infection loads to identify resistance levels of sesame genotypes against phyllody phytoplasma disease. PMID:27195795

  6. Multiplex Real-Time qPCR Assay for Simultaneous and Sensitive Detection of Phytoplasmas in Sesame Plants and Insect Vectors.

    PubMed

    Ikten, Cengiz; Ustun, Rustem; Catal, Mursel; Yol, Engin; Uzun, Bulent

    2016-01-01

    Phyllody, a destructive and economically important disease worldwide caused by phytoplasma infections, is characterized by the abnormal development of floral structures into stunted leafy parts and contributes to serious losses in crop plants, including sesame (Sesamum indicum L.). Accurate identification, differentiation, and quantification of phyllody-causing phytoplasmas are essential for effective management of this plant disease and for selection of resistant sesame varieties. In this study, a diagnostic multiplex qPCR assay was developed using TaqMan® chemistry based on detection of the 16S ribosomal RNA gene of phytoplasmas and the 18S ribosomal gene of sesame. Phytoplasma and sesame specific primers and probes labeled with different fluorescent dyes were used for simultaneous amplification of 16SrII and 16SrIX phytoplasmas in a single tube. The multiplex real-time qPCR assay allowed accurate detection, differentiation, and quantification of 16SrII and 16SrIX groups in 109 sesame plant and 92 insect vector samples tested. The assay was found to have a detection sensitivity of 1.8 x 102 and 1.6 x 102 DNA copies for absolute quantification of 16SrII and 16SrIX group phytoplasmas, respectively. Relative quantification was effective and reliable for determination of phyllody phytoplasma DNA amounts normalized to sesame DNA in infected plant tissues. The development of this qPCR assay provides a method for the rapid measurement of infection loads to identify resistance levels of sesame genotypes against phyllody phytoplasma disease. PMID:27195795

  7. Evaluation of the sensitivity of two recently developed STR multiplexes for the analysis of chimerism after haematopoietic stem cell transplantation.

    PubMed

    Odriozola, A; Riancho, J A; Colorado, M; Zarrabeitia, M T

    2013-04-01

    Forensic-oriented kits analysing short tandem repeat (STR) polymorphisms are widely used to determine the proportions of donor and recipient cells after haematopoietic stem cell transplantation. The sensitivity of this technology is crucial for the early detection of relapse and, in consequence, the adjustment of the treatment to enhance donor-origin haematopoiesis in transplant recipients. The objective of this study was to compare the performance of two recently developed STR multiplex kits, AmpFℓSTR(®) Identifiler(®) Plus PCR Amplification Kit (Applied Biosystems) and Investigator™ IDplex(®) (Qiagen), in the analysis of chimerism. Fifteen STR loci were amplified with both kits in 26 peripheral blood samples of transplantated patients showing chimerism. Peak amplitude threshold, detection limit (%DL), per cent donor chimerism and efficacy of each multiplex and STR were determined, and the results with both kits were compared. The %DL and the estimated per cent donor chimerism were similar with both kits. On the other hand, Identifiler(®) Plus kit allowed chimerism identification only in 24 (92%) of the 26 cases with chimerism detected by using the Investigator™ IDplex(®) when only 'type 5' allelic constellations (i.e. without potential interference by stutter peaks) were taken into account. However, IDplex(®) efficacy was somewhat lower than that of Identifiler Plus when only the most informative loci (D2S1338, D21S11, D18S51 and FGA) were considered. Therefore, although each system had some particular advantages and disadvantages, overall both STR multiplexes showed similar performance in qualitative and quantitative chimerism analysis.

  8. Nano metal-organic framework (NMOF)-based strategies for multiplexed microRNA detection in solution and living cancer cells

    NASA Astrophysics Data System (ADS)

    Wu, Yafeng; Han, Jianyu; Xue, Peng; Xu, Rong; Kang, Yuejun

    2015-01-01

    MiRNAs are an emerging type of biomarker for diagnostics and prognostics. A reliable sensing strategy that can monitor miRNA expression in living cancer cells would be critical in view of its extensive advantages for fundamental research related to miRNA-associated bioprocesses and biomedical applications. Conventional miRNA sensing methods include northern blot, microarrays and real-time quantitative PCR. However, none of them is able to monitor miRNA levels expressed in living cancer cells in a real-time fashion. Some fluorescennt biosensors developed recently from carbon nanomaterials, such as single-walled carbon nanotubes (SWNTs), graphene oxide (GO), and carbon nanoparticles, have been successfully used for assaying miRNA in vitro; however the preparation processes are often expensive, complicated and time-consuming, which have motivated the research on other substitute and novel materials. Herein we present a novel sensing strategy based on peptide nucleic acid (PNA) probes labeled with fluorophores and conjugated with an NMOF vehicle to monitor multiplexed miRNAs in living cancer cells. The NMOF works as a fluorescence quencher of the labelled PNA that is firmly bound with the metal center. In the presence of a target miRNA, PNA is hybridized and released from the NMOF leading to the recovery of fluorescence. This miRNA sensor not only enables the quantitative and highly specific detection of multiplexed miRNAs in living cancer cells, but it also allows the precise and in situ monitoring of the spatiotemporal changes of miRNA expression.MiRNAs are an emerging type of biomarker for diagnostics and prognostics. A reliable sensing strategy that can monitor miRNA expression in living cancer cells would be critical in view of its extensive advantages for fundamental research related to miRNA-associated bioprocesses and biomedical applications. Conventional miRNA sensing methods include northern blot, microarrays and real-time quantitative PCR. However, none of

  9. Simple, multiplexed, PCR-based barcoding of DNA enables sensitive mutation detection in liquid biopsies using sequencing.

    PubMed

    Ståhlberg, Anders; Krzyzanowski, Paul M; Jackson, Jennifer B; Egyud, Matthew; Stein, Lincoln; Godfrey, Tony E

    2016-06-20

    Detection of cell-free DNA in liquid biopsies offers great potential for use in non-invasive prenatal testing and as a cancer biomarker. Fetal and tumor DNA fractions however can be extremely low in these samples and ultra-sensitive methods are required for their detection. Here, we report an extremely simple and fast method for introduction of barcodes into DNA libraries made from 5 ng of DNA. Barcoded adapter primers are designed with an oligonucleotide hairpin structure to protect the molecular barcodes during the first rounds of polymerase chain reaction (PCR) and prevent them from participating in mis-priming events. Our approach enables high-level multiplexing and next-generation sequencing library construction with flexible library content. We show that uniform libraries of 1-, 5-, 13- and 31-plex can be generated. Utilizing the barcodes to generate consensus reads for each original DNA molecule reduces background sequencing noise and allows detection of variant alleles below 0.1% frequency in clonal cell line DNA and in cell-free plasma DNA. Thus, our approach bridges the gap between the highly sensitive but specific capabilities of digital PCR, which only allows a limited number of variants to be analyzed, with the broad target capability of next-generation sequencing which traditionally lacks the sensitivity to detect rare variants. PMID:27060140

  10. Simple, multiplexed, PCR-based barcoding of DNA enables sensitive mutation detection in liquid biopsies using sequencing

    PubMed Central

    Ståhlberg, Anders; Krzyzanowski, Paul M.; Jackson, Jennifer B.; Egyud, Matthew; Stein, Lincoln; Godfrey, Tony E.

    2016-01-01

    Detection of cell-free DNA in liquid biopsies offers great potential for use in non-invasive prenatal testing and as a cancer biomarker. Fetal and tumor DNA fractions however can be extremely low in these samples and ultra-sensitive methods are required for their detection. Here, we report an extremely simple and fast method for introduction of barcodes into DNA libraries made from 5 ng of DNA. Barcoded adapter primers are designed with an oligonucleotide hairpin structure to protect the molecular barcodes during the first rounds of polymerase chain reaction (PCR) and prevent them from participating in mis-priming events. Our approach enables high-level multiplexing and next-generation sequencing library construction with flexible library content. We show that uniform libraries of 1-, 5-, 13- and 31-plex can be generated. Utilizing the barcodes to generate consensus reads for each original DNA molecule reduces background sequencing noise and allows detection of variant alleles below 0.1% frequency in clonal cell line DNA and in cell-free plasma DNA. Thus, our approach bridges the gap between the highly sensitive but specific capabilities of digital PCR, which only allows a limited number of variants to be analyzed, with the broad target capability of next-generation sequencing which traditionally lacks the sensitivity to detect rare variants. PMID:27060140

  11. Identification of Sensitive Serum microRNA Biomarkers for Radiation Biodosimetry

    PubMed Central

    Jacob, Naduparambil Korah; Cooley, James V.; Yee, Tamara N.; Jacob, Jidhin; Alder, Hansjuerg; Wickramasinghe, Priyankara; Maclean, Kirsteen H.; Chakravarti, Arnab

    2013-01-01

    Exposure to ionizing radiation through environmental, occupational or a nuclear reactor accident such as the recent Fukushima Daiichi incident often results in major consequences to human health. The injury caused by radiation can manifest as acute radiation syndromes within weeks in organs with proliferating cells such as hematopoietic and gastrointestinal systems. Cancers, fibrosis and degenerative diseases are also reported in organs with differentiated cells, months or years later. Studies conducted on atom bomb survivors, nuclear reactor workers and animal models have shown a direct correlation of these effects with the absorbed dose. Physical dosimeters and the available radio-responsive biologics in body fluids, whose responses are rather indirect, have limitations to accurately evaluate the extent of post exposure damage. We have used an amplification-free, hybridization based quantitative assay utilizing the nCounter multiplex platform developed by nanoString Technologies to compare the levels of over 600 miRNAs in serum from mice irradiated at a range of 1 to 12 Gy at 24 and 48 hr time points. Development of a novel normalization strategy using multiple spike-in oligonucleotides allowed accurate measurement of radiation dose and time dependent changes in serum miRNAs. The response of several evolutionarily conserved miRNAs abundant in serum, were found to be robust and sensitive in the dose range relevant for medical triage and in patients who receive total body radiation as preparative regimen for bone marrow transplantation. Notably, miRNA-150, abundant in lymphocytes, exhibited a dose and time dependent decrease in serum, which we propose as a sensitive marker indicative of lymphocyte depletion and bone marrow damage. Our study has identified several markers useful for evaluation of an individual’s response by minimally invasive methods, relevant to triage in case of a radiation accident and evaluation of toxicity and response during and after

  12. Identification of sensitive serum microRNA biomarkers for radiation biodosimetry.

    PubMed

    Jacob, Naduparambil Korah; Cooley, James V; Yee, Tamara N; Jacob, Jidhin; Alder, Hansjuerg; Wickramasinghe, Priyankara; Maclean, Kirsteen H; Chakravarti, Arnab

    2013-01-01

    Exposure to ionizing radiation through environmental, occupational or a nuclear reactor accident such as the recent Fukushima Daiichi incident often results in major consequences to human health. The injury caused by radiation can manifest as acute radiation syndromes within weeks in organs with proliferating cells such as hematopoietic and gastrointestinal systems. Cancers, fibrosis and degenerative diseases are also reported in organs with differentiated cells, months or years later. Studies conducted on atom bomb survivors, nuclear reactor workers and animal models have shown a direct correlation of these effects with the absorbed dose. Physical dosimeters and the available radio-responsive biologics in body fluids, whose responses are rather indirect, have limitations to accurately evaluate the extent of post exposure damage. We have used an amplification-free, hybridization based quantitative assay utilizing the nCounter multiplex platform developed by nanoString Technologies to compare the levels of over 600 miRNAs in serum from mice irradiated at a range of 1 to 12 Gy at 24 and 48 hr time points. Development of a novel normalization strategy using multiple spike-in oligonucleotides allowed accurate measurement of radiation dose and time dependent changes in serum miRNAs. The response of several evolutionarily conserved miRNAs abundant in serum, were found to be robust and sensitive in the dose range relevant for medical triage and in patients who receive total body radiation as preparative regimen for bone marrow transplantation. Notably, miRNA-150, abundant in lymphocytes, exhibited a dose and time dependent decrease in serum, which we propose as a sensitive marker indicative of lymphocyte depletion and bone marrow damage. Our study has identified several markers useful for evaluation of an individual's response by minimally invasive methods, relevant to triage in case of a radiation accident and evaluation of toxicity and response during and after

  13. Multiplexed, Quantitative Workflow for Sensitive Biomarker Discovery in Plasma Yields Novel Candidates for Early Myocardial Injury*

    PubMed Central

    Keshishian, Hasmik; Burgess, Michael W.; Gillette, Michael A.; Mertins, Philipp; Clauser, Karl R.; Mani, D. R.; Kuhn, Eric W.; Farrell, Laurie A.; Gerszten, Robert E.; Carr, Steven A.

    2015-01-01

    We have developed a novel plasma protein analysis platform with optimized sample preparation, chromatography, and MS analysis protocols. The workflow, which utilizes chemical isobaric mass tag labeling for relative quantification of plasma proteins, achieves far greater depth of proteome detection and quantification while simultaneously having increased sample throughput than prior methods. We applied the new workflow to a time series of plasma samples from patients undergoing a therapeutic, “planned” myocardial infarction for hypertrophic cardiomyopathy, a unique human model in which each person serves as their own biologic control. Over 5300 proteins were confidently identified in our experiments with an average of 4600 proteins identified per sample (with two or more distinct peptides identified per protein) using iTRAQ four-plex labeling. Nearly 3400 proteins were quantified in common across all 16 patient samples. Compared with a previously published label-free approach, the new method quantified almost fivefold more proteins/sample and provided a six- to nine-fold increase in sample analysis throughput. Moreover, this study provides the largest high-confidence plasma proteome dataset available to date. The reliability of relative quantification was also greatly improved relative to the label-free approach, with measured iTRAQ ratios and temporal trends correlating well with results from a 23-plex immunoMRM (iMRM) assay containing a subset of the candidate proteins applied to the same patient samples. The functional importance of improved detection and quantification was reflected in a markedly expanded list of significantly regulated proteins that provided many new candidate biomarker proteins. Preliminary evaluation of plasma sample labeling with TMT six-plex and ten-plex reagents suggests that even further increases in multiplexing of plasma analysis are practically achievable without significant losses in depth of detection relative to iTRAQ four

  14. RNase protection assays and RNA gel blots: a direct comparison of sensitivity.

    PubMed

    Higgs, D C; Colbert, J T

    1992-01-01

    RNase protection assays are commonly thought to be a more sensitive means of detecting and quantitating specific mRNAs than are RNA gel blots (Northern blots). We have directly compared the sensitivity of these two approaches by assaying for known amounts of in vitro synthesized beta-glucuronidase mRNA. With the probes and protocols employed here, the ability to detect a specific mRNA was similar whether RNase protection or RNA gel blot analyses were performed.

  15. Sensitive and specific detection of miRNA using an isothermal exponential amplification method using fluorescence-labeled LNA/DNA chimera primers.

    PubMed

    Huang, Jun-Fu; Zhao, Na; Xu, Han-Qing; Xia, Han; Wei, Kun; Fu, Wei-Ling; Huang, Qing

    2016-10-01

    MicroRNAs (miRNAs) are currently considered as potential biomarkers for various human diseases. In the present study, miRNA-triggered real-time fluorescent isothermal reaction with exponential amplification (ReFIRE) with or without Thermus aquaticus MutS (Taq MutS) was developed to analyze miRNAs using DNA polymerase, a nicking endonuclease, and fluorescently labeled primers. In the absence of Taq MutS, the ReFIRE system permitted the detection of 100 ymol of targeted miRNA in 80 min. However, this system enabled limited differentiation between homologous miRNA family members. Upon addition of Taq MutS to the ReFIRE system, non-specific amplification generated from the mishybridization between primers and primer dimers or primers and the template duplex was eliminated. The addition of Taq MutS enabled the ultrasensitive detection of as little as 10 ymol of targeted miRNAs in 50 min, which corresponds to less than 10 copies of miRNAs in a total volume of 20 μl. Additionally, the assay exhibited a dynamic range of up to 12 orders of magnitude. The ReFIRE system also showed high specificity, enabling differentiation between homologous miRNA family members exhibiting only single-base differences. The sensitivity, specificity, and dynamic range associated with this system were greater than most currently available miRNA isothermal amplification assays. Moreover, when target-specific primers were labeled with different fluorescent reporters, multiplex analysis was easily performed in a single tube, permitting accurate normalization of miRNA expression. This simple, fast, ultrasensitive, highly specific, and easy-to-multiplex method could significantly contribute to research investigations pertaining to the biological roles of miRNA, as well as clinical diagnosis of various diseases that involve miRNA disruptions. Graphical Abstract The principle of ReFIRE system.

  16. Sensitive and specific detection of miRNA using an isothermal exponential amplification method using fluorescence-labeled LNA/DNA chimera primers.

    PubMed

    Huang, Jun-Fu; Zhao, Na; Xu, Han-Qing; Xia, Han; Wei, Kun; Fu, Wei-Ling; Huang, Qing

    2016-10-01

    MicroRNAs (miRNAs) are currently considered as potential biomarkers for various human diseases. In the present study, miRNA-triggered real-time fluorescent isothermal reaction with exponential amplification (ReFIRE) with or without Thermus aquaticus MutS (Taq MutS) was developed to analyze miRNAs using DNA polymerase, a nicking endonuclease, and fluorescently labeled primers. In the absence of Taq MutS, the ReFIRE system permitted the detection of 100 ymol of targeted miRNA in 80 min. However, this system enabled limited differentiation between homologous miRNA family members. Upon addition of Taq MutS to the ReFIRE system, non-specific amplification generated from the mishybridization between primers and primer dimers or primers and the template duplex was eliminated. The addition of Taq MutS enabled the ultrasensitive detection of as little as 10 ymol of targeted miRNAs in 50 min, which corresponds to less than 10 copies of miRNAs in a total volume of 20 μl. Additionally, the assay exhibited a dynamic range of up to 12 orders of magnitude. The ReFIRE system also showed high specificity, enabling differentiation between homologous miRNA family members exhibiting only single-base differences. The sensitivity, specificity, and dynamic range associated with this system were greater than most currently available miRNA isothermal amplification assays. Moreover, when target-specific primers were labeled with different fluorescent reporters, multiplex analysis was easily performed in a single tube, permitting accurate normalization of miRNA expression. This simple, fast, ultrasensitive, highly specific, and easy-to-multiplex method could significantly contribute to research investigations pertaining to the biological roles of miRNA, as well as clinical diagnosis of various diseases that involve miRNA disruptions. Graphical Abstract The principle of ReFIRE system. PMID:27485624

  17. Simultaneous detection and identification of four cherry viruses by two step multiplex RT-PCR with an internal control of plant nad5 mRNA.

    PubMed

    Noorani, Md Salik; Awasthi, Prachi; Sharma, Maheshwar Prasad; Ram, Raja; Zaidi, Aijaz Asgar; Hallan, Vipin

    2013-10-01

    A multiplex reverse transcription-polymerase chain reaction (mRT-PCR) was developed and standardized for the simultaneous detection of four cherry viruses: Cherry virus A (CVA, Genus; Capillovirus), Cherry necrotic rusty mottle virus (CNRMV, unassigned species of the Betaflexiviridae), Little cherry virus 1 (LChV-1, Genus; Closterovirus) and Prunus necrotic ringspot virus (PNRSV, Genus; Ilarvirus) with nad5 as plant internal control. A reliable and quick method for total plant RNA extraction from pome and stone fruit trees was also developed. To minimize primer dimer formation, a single antisense primer for CVA and CNRMV was used. A mixture of random hexamer and oligo (dT) primer was used for cDNA synthesis, which was highly suited and economic for multiplexing. All four viruses were detected successfully by mRT-PCR in artificially created viral RNA mixture and field samples of sweet cherry. The identity of the viruses was confirmed by sequencing. The assay could detect above viruses in diluted cDNA (10(-4)) and RNA (10(-3), except PNRSV which was detected only till ten times lesser dilution). The developed mRT-PCR will not only be useful for the detection of viruses from single or multiple infections of sweet cherry plants but also for other stone and pome fruits. The developed method will be therefore quite helpful for virus indexing, plant quarantine and certification programs. This is the first report for the simultaneous detection of four cherry viruses by mRT-PCR.

  18. Deciphering Noncoding RNA and Chromatin Interactions: Multiplex Chromatin Interaction Analysis by Paired-End Tag Sequencing (mChIA-PET).

    PubMed

    Choy, Jocelyn; Fullwood, Melissa J

    2017-01-01

    Genomic DNA is dynamically associated with protein factors and folded to form chromatin fibers. The 3-dimensional (3D) configuration of the chromatin will enable the distal genetic elements to come into close proximity, allowing transcriptional regulation. Noncoding RNA can mediate the 3D structure of chromatin. Chromatin Interaction Analysis by Paired-End Tag Sequencing (ChIA-PET) is a valuable and powerful technique in molecular biology which allows the study of unbiased, genome-wide de novo chromatin interactions with paired-end tags. Here, we describe the standard version of ChIA-PET and a Multiplex ChIA-PET version. PMID:27662871

  19. Multiplexed Digital mRNA Profiling of the Inflammatory Response in the West Nile Swiss Webster Mouse Model

    PubMed Central

    Peña, José; Plante, Jessica A.; Carillo, Alda Celena; Roberts, Kimberly K.; Smith, Jennifer K.; Juelich, Terry L.; Beasley, David W. C.; Freiberg, Alexander N.; Labute, Montiago X.; Naraghi-Arani, Pejman

    2014-01-01

    Background and purpose The ability to track changes in gene expression following viral infection is paramount to understanding viral pathogenesis. This study was undertaken to evaluate the nCounter, a high throughput digital gene expression system, as a means to better understand West Nile virus (WNV) dissemination and the inflammatory response against WNV in the outbred Swiss Webster (SW) mouse model over the course of infection. Methodology The nCounter Mouse Inflammation gene expression kit containing 179 inflammation related genes was used to analyze gene expression changes in multiple tissues over a nine day course of infection in SW mice following intraperitoneal injection with WNV. Protein expression levels for a subset of these cytokine/chemokine genes were determined using a multiplex protein detection system (BioPlex) and comparisons of protein/RNA expression levels made. Results Expression analysis of spleen, lung, liver, kidney and brain of SW mice infected with WNV revealed that Cxcl10 and Il12b are differentially expressed in all tissues tested except kidney. Data stratification of positively confirmed infected (WNV (+)) versus non-infected (WNV (−) tissues allowed differentiation of the systemic inflammatory gene response from tissue-specific responses arising from WNV infection. Significant (p<0.05) decrease in C3ar1 was found in WNV (−) spleen. Il23a was significantly upregulated, while Il10rb was down-regulated in WNV (−) lung. Il3 and Mbl2 were down-regulated in WNV (−) liver. In WNV (+) livers, Stat1, Tlr2, chemokines Cxcl1, Cxcl3, Cxcl9, Cxcl10, cytokines Il6, Il18, cytokine-related gene Il1r and cytokine agonist Ilrn were significantly upregulated. In WNV (−) brain tissues, Csf2 and Cxcl10 were significantly upregulated. Similar gene and protein expression kinetics were found for Ccl2, Ccl3, Ccl4 and Ccl5 and correlated with the presence of infectious virus. In summary, the utility of the nCounter platform for rapid identification of

  20. Fluorescent Protein Nanowire-Mediated Protein Microarrays for Multiplexed and Highly Sensitive Pathogen Detection.

    PubMed

    Men, Dong; Zhou, Juan; Li, Wei; Leng, Yan; Chen, Xinwen; Tao, Shengce; Zhang, Xian-En

    2016-07-13

    Protein microarrays are powerful tools for high-throughput and simultaneous detection of different target molecules in complex biological samples. However, the sensitivity of conventional fluorescence-labeling protein detection methods is limited by the availability of signal molecules for binding to the target molecule. Here, we built a multifunctional fluorescent protein nanowire (FNw) by harnessing self-assembly of yeast amyloid protein. The FNw integrated a large number of fluorescent molecules, thereby enhancing the fluorescent signal output in target detection. The FNw was then combined with protein microarray technology to detect proteins derived from two pathogens, including influenza virus (hemagglutinin 1, HA1) and human immunodeficiency virus (p24 and gp120). The resulting detection sensitivity achieved a 100-fold improvement over a commercially available detection reagent. PMID:27315221

  1. Silicon on-chip bandpass filters for the multiplexing of high sensitivity photonic crystal microcavity biosensors

    SciTech Connect

    Yan, Hai Zou, Yi; Yang, Chun-Ju; Chakravarty, Swapnajit; Wang, Zheng; Tang, Naimei; Chen, Ray T.; Fan, Donglei

    2015-03-23

    A method for the dense integration of high sensitivity photonic crystal (PC) waveguide based biosensors is proposed and experimentally demonstrated on a silicon platform. By connecting an additional PC waveguide filter to a PC microcavity sensor in series, a transmission passband is created, containing the resonances of the PC microcavity for sensing purpose. With proper engineering of the passband, multiple high sensitivity PC microcavity sensors can be integrated into microarrays and be interrogated simultaneously between a single input and a single output port. The concept was demonstrated with a 2-channel L55 PC biosensor array containing PC waveguide filters. The experiment showed that the sensors on both channels can be monitored simultaneously from a single output spectrum. Less than 3 dB extra loss for the additional PC waveguide filter is observed.

  2. High-sensitivity high-throughput chip based biosensor array for multiplexed detection of heavy metals

    NASA Astrophysics Data System (ADS)

    Yan, Hai; Tang, Naimei; Jairo, Grace A.; Chakravarty, Swapnajit; Blake, Diane A.; Chen, Ray T.

    2016-03-01

    Heavy metal ions released into the environment from industrial processes lead to various health hazards. We propose an on-chip label-free detection approach that allows high-sensitivity and high-throughput detection of heavy metals. The sensing device consists of 2-dimensional photonic crystal microcavities that are combined by multimode interferometer to form a sensor array. We experimentally demonstrate the detection of cadmium-chelate conjugate with concentration as low as 5 parts-per-billion (ppb).

  3. Sensitive simultaneous detection of seven sexually transmitted agents in semen by multiplex-PCR and of HPV by single PCR.

    PubMed

    Gimenes, Fabrícia; Medina, Fabiana Soares; Abreu, André Luelsdorf Pimenta de; Irie, Mary Mayumi Taguti; Esquiçati, Isis Baroni; Malagutti, Natália; Vasconcellos, Vinícius Rodrigo Bulla; Discacciati, Michele Garcia; Bonini, Marcelo Gialluisi; Maria-Engler, Silvya Stuchi; Consolaro, Marcia Edilaine Lopes

    2014-01-01

    Sexually transmitted diseases (STDs) may impair sperm parameters and functions thereby promoting male infertility. To date limited molecular studies were conducted to evaluate the frequency and type of such infections in semen Thus, we aimed at conceiving and validating a multiplex PCR (M-PCR) assay for the simultaneous detection of the following STD pathogens in semen: Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma genitalium, Trichomonas vaginalis, Herpes virus simplex (HSV) -1 and -2, and Treponema pallidum; We also investigated the potential usefulness of this M-PCR assay in screening programs for semen pathogens. In addition, we aimed: to detect human Papillomavirus (HPV) and genotypes by single PCR (sPCR) in the same semen samples; to determine the prevalence of the seven STDs, HPV and co-infections; to assess the possibility that these infections affect semen parameters and thus fertility. The overall validation parameters of M-PCR were extremely high including agreement (99.2%), sensitivity (100.00%), specificity (99.70%), positive (96.40%) and negative predictive values (100.00%) and accuracy (99.80%). The prevalence of STDs was very high (55.3%). Furthermore, associations were observed between STDs and changes in semen parameters, highlighting the importance of STD detection in semen. Thus, this M-PCR assay has great potential for application in semen screening programs for pathogens in infertility and STD clinics and in sperm banks.

  4. Sets of RNA Repeated Tags and Hybridization-Sensitive Fluorescent Probes for Distinct Images of RNA in a Living Cell

    PubMed Central

    Kubota, Takeshi; Ikeda, Shuji; Yanagisawa, Hiroyuki; Yuki, Mizue; Okamoto, Akimitsu

    2010-01-01

    Background Imaging the behavior of RNA in a living cell is a powerful means for understanding RNA functions and acquiring spatiotemporal information in a single cell. For more distinct RNA imaging in a living cell, a more effective chemical method to fluorescently label RNA is now required. In addition, development of the technology labeling with different colors for different RNA would make it easier to analyze plural RNA strands expressing in a cell. Methodology/Principal Findings Tag technology for RNA imaging in a living cell has been developed based on the unique chemical functions of exciton-controlled hybridization-sensitive oligonucleotide (ECHO) probes. Repetitions of selected 18-nucleotide RNA tags were incorporated into the mRNA 3′-UTR. Pairs with complementary ECHO probes exhibited hybridization-sensitive fluorescence emission for the mRNA expressed in a living cell. The mRNA in a nucleus was detected clearly as fluorescent puncta, and the images of the expression of two mRNAs were obtained independently and simultaneously with two orthogonal tag–probe pairs. Conclusions/Significance A compact and repeated label has been developed for RNA imaging in a living cell, based on the photochemistry of ECHO probes. The pairs of an 18-nt RNA tag and the complementary ECHO probes are highly thermostable, sequence-specifically emissive, and orthogonal to each other. The nucleotide length necessary for one tag sequence is much shorter compared with conventional tag technologies, resulting in easy preparation of the tag sequences with a larger number of repeats for more distinct RNA imaging. PMID:20885944

  5. Quenching of Unincorporated Amplification Signal Reporters in Reverse-Transcription Loop-Mediated Isothermal Amplification Enabling Bright, Single-Step, Closed-Tube, and Multiplexed Detection of RNA Viruses.

    PubMed

    Ball, Cameron S; Light, Yooli K; Koh, Chung-Yan; Wheeler, Sarah S; Coffey, Lark L; Meagher, Robert J

    2016-04-01

    Reverse-transcription-loop-mediated isothermal amplification (RT-LAMP) has frequently been proposed as an enabling technology for simplified diagnostic tests for RNA viruses. However, common detection techniques used for LAMP and RT-LAMP have drawbacks, including poor discrimination capability, inability to multiplex targets, high rates of false positives, and (in some cases) the requirement of opening reaction tubes postamplification. Here, we present a simple technique that allows closed-tube, target-specific detection, based on inclusion of a dye-labeled primer that is incorporated into a target-specific amplicon if the target is present. A short, complementary quencher hybridizes to unincorporated primer upon cooling down at the end of the reaction, thereby quenching fluorescence of any unincorporated primer. Our technique, which we term QUASR (for quenching of unincorporated amplification signal reporters, read "quasar"), does not significantly reduce the amplification efficiency or sensitivity of RT-LAMP. Equipped with a simple LED excitation source and a colored plastic gel filter, the naked eye or a camera can easily discriminate between positive and negative QUASR reactions, which produce a difference in signal of approximately 10:1 without background subtraction. We demonstrate that QUASR detection is compatible with complex sample matrices such as human blood, using a novel LAMP primer set for bacteriophage MS2 (a model RNA virus particle). Furthermore, we demonstrate single-tube duplex detection of West Nile virus (WNV) and chikungunya virus (CHIKV) RNA.

  6. Quenching of Unincorporated Amplification Signal Reporters in Reverse-Transcription Loop-Mediated Isothermal Amplification Enabling Bright, Single-Step, Closed-Tube, and Multiplexed Detection of RNA Viruses.

    PubMed

    Ball, Cameron S; Light, Yooli K; Koh, Chung-Yan; Wheeler, Sarah S; Coffey, Lark L; Meagher, Robert J

    2016-04-01

    Reverse-transcription-loop-mediated isothermal amplification (RT-LAMP) has frequently been proposed as an enabling technology for simplified diagnostic tests for RNA viruses. However, common detection techniques used for LAMP and RT-LAMP have drawbacks, including poor discrimination capability, inability to multiplex targets, high rates of false positives, and (in some cases) the requirement of opening reaction tubes postamplification. Here, we present a simple technique that allows closed-tube, target-specific detection, based on inclusion of a dye-labeled primer that is incorporated into a target-specific amplicon if the target is present. A short, complementary quencher hybridizes to unincorporated primer upon cooling down at the end of the reaction, thereby quenching fluorescence of any unincorporated primer. Our technique, which we term QUASR (for quenching of unincorporated amplification signal reporters, read "quasar"), does not significantly reduce the amplification efficiency or sensitivity of RT-LAMP. Equipped with a simple LED excitation source and a colored plastic gel filter, the naked eye or a camera can easily discriminate between positive and negative QUASR reactions, which produce a difference in signal of approximately 10:1 without background subtraction. We demonstrate that QUASR detection is compatible with complex sample matrices such as human blood, using a novel LAMP primer set for bacteriophage MS2 (a model RNA virus particle). Furthermore, we demonstrate single-tube duplex detection of West Nile virus (WNV) and chikungunya virus (CHIKV) RNA. PMID:26980448

  7. Microfluidic based multiplex qRT-PCR identifies diagnostic and prognostic microRNA signatures in sera of prostate cancer patients

    PubMed Central

    Moltzahn, Felix; Olshen, Adam B.; Baehner, Lauren; Peek, Andrew; Fong, Lawrence; Stöppler, Hubert; Simko, Jeffry; Hilton, Joan F.; Carroll, Peter; Blelloch, Robert

    2010-01-01

    Recent prostate specific antigen (PSA) based screening trials indicate an urgent need for novel and non-invasive biomarker identification strategies to improve the prediction of prostate cancer behavior. Non-coding microRNAs (miRNAs) in the serum and plasma have been shown to have potential as non-invasive markers for physiological and pathological conditions. To identify serum miRNAs that diagnose and correlate with prognosis of prostate cancer, we developed a multiplex quantitative reverse transcription PCR (qRT-PCR) method involving purification of multiplex PCR products followed by uniplex analysis on a microfluidics chip to evaluate 384 human miRNAs. Using Dgcr8 and Dicer knockout (small RNA - deficient) mouse ES cells (mESC) as the benchmark, we confirmed the validity of our technique, while uncovering a significant lack of accuracy in previously published methods. Profiling 48 sera from healthy men and untreated prostate cancer patients with differing CAPRA (Cancer of the Prostate Risk Assessment) scores, we identified miRNA signatures that allow to diagnose cancer patients and correlate with prognosis. These serum signatures include oncogenic and tumor suppressive miRNAs suggesting functional roles in prostate cancer progression. PMID:21098088

  8. Rapid and Sensitive Multiplex Detection of Burkholderia pseudomallei-Specific Antibodies in Melioidosis Patients Based on a Protein Microarray Approach

    PubMed Central

    Kohler, Christian; Dunachie, Susanna J.; Müller, Elke; Kohler, Anne; Jenjaroen, Kemajittra; Teparrukkul, Prapit; Baier, Vico; Ehricht, Ralf; Steinmetz, Ivo

    2016-01-01

    Background The environmental bacterium Burkholderia pseudomallei causes the infectious disease melioidosis with a high case-fatality rate in tropical and subtropical regions. Direct pathogen detection can be difficult, and therefore an indirect serological test which might aid early diagnosis is desirable. However, current tests for antibodies against B. pseudomallei, including the reference indirect haemagglutination assay (IHA), lack sensitivity, specificity and standardization. Consequently, serological tests currently do not play a role in the diagnosis of melioidosis in endemic areas. Recently, a number of promising diagnostic antigens have been identified, but a standardized, easy-to-perform clinical laboratory test for sensitive multiplex detection of antibodies against B. pseudomallei is still lacking. Methods and Principal Findings In this study, we developed and validated a protein microarray which can be used in a standard 96-well format. Our array contains 20 recombinant and purified B. pseudomallei proteins, previously identified as serodiagnostic candidates in melioidosis. In total, we analyzed 196 sera and plasmas from melioidosis patients from northeast Thailand and 210 negative controls from melioidosis-endemic and non-endemic regions. Our protein array clearly discriminated between sera from melioidosis patients and controls with a specificity of 97%. Importantly, the array showed a higher sensitivity than did the IHA in melioidosis patients upon admission (cut-off IHA titer ≥1:160: IHA 57.3%, protein array: 86.7%; p = 0.0001). Testing of sera from single patients at 0, 12 and 52 weeks post-admission revealed that protein antigens induce either a short- or long-term antibody response. Conclusions Our protein array provides a standardized, rapid, easy-to-perform test for the detection of B. pseudomallei-specific antibody patterns. Thus, this system has the potential to improve the serodiagnosis of melioidosis in clinical settings. Moreover, our

  9. Antagonistic functions between the RNA chaperone Hfq and an sRNA regulate sensitivity to the antibiotic colicin.

    PubMed

    Salvail, Hubert; Caron, Marie-Pier; Bélanger, Justine; Massé, Eric

    2013-10-16

    The RNA chaperone Hfq is a key regulator of the function of small RNAs (sRNAs). Hfq has been shown to facilitate sRNAs binding to target mRNAs and to directly regulate translation through the action of sRNAs. Here, we present evidence that Hfq acts as the repressor of cirA mRNA translation in the absence of sRNA. Hfq binding to cirA prevents translation initiation, which correlates with cirA mRNA instability. In contrast, RyhB pairing to cirA mRNA promotes changes in RNA structure that displace Hfq, thereby allowing efficient translation as well as mRNA stabilization. Because CirA is a receptor for the antibiotic colicin Ia, in addition to acting as an Fur (Ferric Uptake Regulator)-regulated siderophore transporter, translational activation of cirA mRNA by RyhB promotes colicin sensitivity under conditions of iron starvation. Altogether, these results indicate that Fur and RyhB modulate an unexpected feed-forward loop mechanism related to iron physiology and colicin sensitivity. PMID:24065131

  10. Antagonistic functions between the RNA chaperone Hfq and an sRNA regulate sensitivity to the antibiotic colicin

    PubMed Central

    Salvail, Hubert; Caron, Marie-Pier; Bélanger, Justine; Massé, Eric

    2013-01-01

    The RNA chaperone Hfq is a key regulator of the function of small RNAs (sRNAs). Hfq has been shown to facilitate sRNAs binding to target mRNAs and to directly regulate translation through the action of sRNAs. Here, we present evidence that Hfq acts as the repressor of cirA mRNA translation in the absence of sRNA. Hfq binding to cirA prevents translation initiation, which correlates with cirA mRNA instability. In contrast, RyhB pairing to cirA mRNA promotes changes in RNA structure that displace Hfq, thereby allowing efficient translation as well as mRNA stabilization. Because CirA is a receptor for the antibiotic colicin Ia, in addition to acting as an Fur (Ferric Uptake Regulator)-regulated siderophore transporter, translational activation of cirA mRNA by RyhB promotes colicin sensitivity under conditions of iron starvation. Altogether, these results indicate that Fur and RyhB modulate an unexpected feed-forward loop mechanism related to iron physiology and colicin sensitivity. PMID:24065131

  11. mCAL: A New Approach for Versatile Multiplex Action of Cas9 Using One sgRNA and Loci Flanked by a Programmed Target Sequence

    PubMed Central

    Finnigan, Gregory C.; Thorner, Jeremy

    2016-01-01

    Genome editing exploiting CRISPR/Cas9 has been adopted widely in academia and in the biotechnology industry to manipulate DNA sequences in diverse organisms. Molecular engineering of Cas9 itself and its guide RNA, and the strategies for using them, have increased efficiency, optimized specificity, reduced inappropriate off-target effects, and introduced modifications for performing other functions (transcriptional regulation, high-resolution imaging, protein recruitment, and high-throughput screening). Moreover, Cas9 has the ability to multiplex, i.e., to act at different genomic targets within the same nucleus. Currently, however, introducing concurrent changes at multiple loci involves: (i) identification of appropriate genomic sites, especially the availability of suitable PAM sequences; (ii) the design, construction, and expression of multiple sgRNA directed against those sites; (iii) potential difficulties in altering essential genes; and (iv) lingering concerns about “off-target” effects. We have devised a new approach that circumvents these drawbacks, as we demonstrate here using the yeast Saccharomyces cerevisiae. First, any gene(s) of interest are flanked upstream and downstream with a single unique target sequence that does not normally exist in the genome. Thereafter, expression of one sgRNA and cotransformation with appropriate PCR fragments permits concomitant Cas9-mediated alteration of multiple genes (both essential and nonessential). The system we developed also allows for maintenance of the integrated, inducible Cas9-expression cassette or its simultaneous scarless excision. Our scheme—dubbed mCAL for “Multiplexing of Cas9 at Artificial Loci”—can be applied to any organism in which the CRISPR/Cas9 methodology is currently being utilized. In principle, it can be applied to install synthetic sequences into the genome, to generate genomic libraries, and to program strains or cell lines so that they can be conveniently (and repeatedly

  12. Primers with 5' flaps improve the efficiency and sensitivity of multiplex PCR assays for the detection of Salmonella and Escherichia coli O157:H7.

    PubMed

    Timmons, Chris; Dobhal, Shefali; Fletcher, Jacqueline; Ma, Li Maria

    2013-04-01

    Foodborne illnesses caused by Salmonella enterica and Escherichia coli O157:H7 are worldwide health concerns. Rapid, sensitive, and robust detection of these pathogens in foods and in clinical and environmental samples is essential for routine food quality testing, effective surveillance, and outbreak investigations. The aim of this study was to evaluate the effect on PCR sensitivity of adding a short, AT-rich overhanging nucleotide sequence (flap) to the 5' end of PCR primers specific for the detection of Salmonella and E. coli O157:H7. Primers targeting the invA gene of Salmonella and the rfbE gene of E. coli O157:H7 were synthesized with or without a 12-bp, AT-rich 5' flap (5'-AATAAATCATAA-3'). Singleplex PCR, multiplex PCR, and real-time PCR sensitivity assays were conducted using purified bacterial genomic DNA and crude cell lysates of bacterial cells. The effect of background flora on detection was evaluated by spiking tomato and jalapeno pepper surface washes with E. coli O157:H7 and Salmonella Saintpaul. When targeting individual pathogens, end-point PCR assays using flap-amended primers were more efficient than nonamended primers, with 20.4 and 23.5% increases in amplicon yield for Salmonella and E. coli O157:H7, respectively. In multiplex PCR assays, a 10- to 100-fold increase in detection sensitivity was observed when the primer flap sequence was incorporated. This improvement in both singleplex and multiplex PCR efficiency and sensitivity can lead to improved Salmonella and E. coli O157:H7 detection.

  13. MicroRNA-21 Increases Proliferation and Cisplatin Sensitivity of Osteosarcoma-Derived Cells.

    PubMed

    Vanas, Vanita; Haigl, Barbara; Stockhammer, Verena; Sutterlüty-Fall, Hedwig

    2016-01-01

    Osteosarcoma is the most common primary bone tumor and poor prognosis for osteosarcoma patients is mainly due to chemotherapy resistance. MicroRNAs are important to maintain pathophysiological mechanisms of cancer and influence cell sensitivity to chemotherapy. In this study, we tested the functions of microRNA-21 for malignant features as well as for drug resistance of osteosarcoma. We used Northern blot to measure microRNA-21 levels in osteosarcoma-derived cell lines. MicroRNA-21 activity was modulated by either expressing a sponge to decrease its activity in an osteosarcoma-derived cell line expressing high levels of microRNA-21 or by introducing pri-microRNA-21 in a cell line with low endogenous levels. Cell migration was determined in a scratch assay and cell proliferation was measured by performing growth curve analysis. Sensitivity of the cells towards chemotherapeutics was investigated by performing cell viability assays and calculating the IC50 values. While cell migration was unaffected by modulated microRNA-21 levels, microRNA-21 inhibition slowed proliferation and exogenously expressed microRNA-21 promoted this process. Modulated microRNA-21 activity failed to effect sensitivity of osteosarcoma-derived cell lines to doxorubicin or methotrexate. Contrarily, reduction of microRNA-21 activity resulted in enhanced resistance towards cisplatin while ectopic expression of microRNA-21 showed the opposite effect. Increased microRNA-21 levels repressed the expression of Sprouty2 and ectopic expression of Sprouty2 was able to largely rescue the observed effects of microRNA-21 in osteosarcoma. In summary, our data indicate that in osteosarcoma microRNA-21 expression is an important component for regulation of cell proliferation and for determining sensitivity to cisplatin. PMID:27513462

  14. MicroRNA-21 Increases Proliferation and Cisplatin Sensitivity of Osteosarcoma-Derived Cells

    PubMed Central

    Vanas, Vanita; Haigl, Barbara; Stockhammer, Verena; Sutterlüty-Fall, Hedwig

    2016-01-01

    Osteosarcoma is the most common primary bone tumor and poor prognosis for osteosarcoma patients is mainly due to chemotherapy resistance. MicroRNAs are important to maintain pathophysiological mechanisms of cancer and influence cell sensitivity to chemotherapy. In this study, we tested the functions of microRNA-21 for malignant features as well as for drug resistance of osteosarcoma. We used Northern blot to measure microRNA-21 levels in osteosarcoma-derived cell lines. MicroRNA-21 activity was modulated by either expressing a sponge to decrease its activity in an osteosarcoma-derived cell line expressing high levels of microRNA-21 or by introducing pri-microRNA-21 in a cell line with low endogenous levels. Cell migration was determined in a scratch assay and cell proliferation was measured by performing growth curve analysis. Sensitivity of the cells towards chemotherapeutics was investigated by performing cell viability assays and calculating the IC50 values. While cell migration was unaffected by modulated microRNA-21 levels, microRNA-21 inhibition slowed proliferation and exogenously expressed microRNA-21 promoted this process. Modulated microRNA-21 activity failed to effect sensitivity of osteosarcoma-derived cell lines to doxorubicin or methotrexate. Contrarily, reduction of microRNA-21 activity resulted in enhanced resistance towards cisplatin while ectopic expression of microRNA-21 showed the opposite effect. Increased microRNA-21 levels repressed the expression of Sprouty2 and ectopic expression of Sprouty2 was able to largely rescue the observed effects of microRNA-21 in osteosarcoma. In summary, our data indicate that in osteosarcoma microRNA-21 expression is an important component for regulation of cell proliferation and for determining sensitivity to cisplatin. PMID:27513462

  15. Interleaved EPI Based fMRI Improved by Multiplexed Sensitivity Encoding (MUSE) and Simultaneous Multi-Band Imaging

    PubMed Central

    Chang, Hing-Chiu; Gaur, Pooja; Chou, Ying-hui; Chu, Mei-Lan; Chen, Nan-kuei

    2014-01-01

    Functional magnetic resonance imaging (fMRI) is a non-invasive and powerful imaging tool for detecting brain activities. The majority of fMRI studies are performed with single-shot echo-planar imaging (EPI) due to its high temporal resolution. Recent studies have demonstrated that, by increasing the spatial-resolution of fMRI, previously unidentified neuronal networks can be measured. However, it is challenging to improve the spatial resolution of conventional single-shot EPI based fMRI. Although multi-shot interleaved EPI is superior to single-shot EPI in terms of the improved spatial-resolution, reduced geometric distortions, and sharper point spread function (PSF), interleaved EPI based fMRI has two main limitations: 1) the imaging throughput is lower in interleaved EPI; 2) the magnitude and phase signal variations among EPI segments (due to physiological noise, subject motion, and B0 drift) are translated to significant in-plane aliasing artifact across the field of view (FOV). Here we report a method that integrates multiple approaches to address the technical limitations of interleaved EPI-based fMRI. Firstly, the multiplexed sensitivity-encoding (MUSE) post-processing algorithm is used to suppress in-plane aliasing artifacts resulting from time-domain signal instabilities during dynamic scans. Secondly, a simultaneous multi-band interleaved EPI pulse sequence, with a controlled aliasing scheme incorporated, is implemented to increase the imaging throughput. Thirdly, the MUSE algorithm is then generalized to accommodate fMRI data obtained with our multi-band interleaved EPI pulse sequence, suppressing both in-plane and through-plane aliasing artifacts. The blood-oxygenation-level-dependent (BOLD) signal detectability and the scan throughput can be significantly improved for interleaved EPI-based fMRI. Our human fMRI data obtained from 3 Tesla systems demonstrate the effectiveness of the developed methods. It is expected that future fMRI studies requiring high

  16. Inhibition of acidic mammalian chitinase by RNA interference suppresses ovalbumin-sensitized allergic asthma.

    PubMed

    Yang, Ching-Jen; Liu, Yu-Kuo; Liu, Chao-Lin; Shen, Chia-Ning; Kuo, Ming-Ling; Su, Chien-Chang; Tseng, Ching-Ping; Yen, Tzu-Chen; Shen, Chia-Rui

    2009-12-01

    Asthma, a chronic helper T cell type 2-mediated inflammatory disease, is characterized by airway hyperresponsiveness and inflammation. Growing evidence suggests that increased expression of acidic mammalian chitinase (AMCase) may play a role in the pathogenesis of asthma. In the present study, we sought to develop an RNA interference approach to suppress allergic asthma in mice through silencing of AMCase expression. Mice sensitized with ovalbumin (OVA) were intratracheally administered a recombinant adeno-associated virus expressing short hairpin RNA (rAAV-shRNA) against AMCase. In OVA-sensitized mice, the development of allergic symptoms was significantly associated with elevated AMCase expression. After administration of rAAV-shRNA, there was a significant reduction of AMCase expression in the lung and in bronchoalveolar lavage fluid (BALF) cells of sensitized mice. Sensitized mice receiving rAAV-shRNA showed a significant improvement in allergic symptoms, including airway hyperresponsiveness (AHR), eosinophil infiltration, eotaxin, interleukin-13 secretion in BALF, and serum OVA-specific IgE level. Our data suggest the hyperexpression of AMCase in asthma can be suppressed by rAAV-mediated shRNA. Silencing AMCase expression by shRNA may be a promising therapeutic strategy in asthma.

  17. Microgels for multiplex and direct fluorescence detection

    NASA Astrophysics Data System (ADS)

    Causa, Filippo; Aliberti, Anna; Cusano, Angela M.; Battista, Edmondo; Netti, Paolo A.

    2015-05-01

    Blood borne oligonucleotides fragments contain useful clinical information whose detection and monitoring represent the new frontier in liquid biopsy as they can transform the current diagnosis procedure. For instance, recent studies have identified a new class of circulating biomarkers such as s miRNAs, and demonstrated that changes in their concentration are closely associated with the development of cancer and other pathologies. However, direct detection of miRNAs in body fluids is particularly challenging and demands high sensitivity -concentration range between atto to femtomolarspecificity, and multiplexing Here we report on engineered multifunctional microgels and innovative probe design for a direct and multiplex detection of relevant clinical miRNAs in fluorescence by single particle assay. Polyethyleneglycol-based microgels have a coreshell architecture with two spectrally encoded fluorescent dyes for multiplex analyses and are endowed with fluorescent probes for miRNA detection. Encoding and detection fluorescence signals are distinguishable by not overlapping emission spectra. Tuneable fluorescence probe conjugation and corresponding emission confinement on single microgel allows for enhanced target detection. Such suspension array has indeed high selectivity and sensitivity with a detection limit of 10-15 M and a dynamic range from 10-9 to 10-15 M. We believe that sensitivity in the fM concentration range, signal background minimization, multiplexed capability and direct measurement of such microgels will translate into diagnostic benefits opening up new roots toward liquid biopsy in the context of point-of-care testing through an easy and fast detection of sensitive diagnostic biomarkers directly in serum.

  18. A combinatorial approach to the repertoire of RNA kissing motifs; towards multiplex detection by switching hairpin aptamers

    PubMed Central

    Durand, Guillaume; Dausse, Eric; Goux, Emma; Fiore, Emmanuelle; Peyrin, Eric; Ravelet, Corinne; Toulmé, Jean-Jacques

    2016-01-01

    Loop–loop (also known as kissing) interactions between RNA hairpins are involved in several mechanisms in both prokaryotes and eukaryotes such as the regulation of the plasmid copy number or the dimerization of retroviral genomes. The stability of kissing complexes relies on loop parameters (base composition, sequence and size) and base combination at the loop–loop helix - stem junctions. In order to identify kissing partners that could be used as regulatory elements or building blocks of RNA scaffolds, we analysed a pool of 5.2 × 106 RNA hairpins with randomized loops. We identified more than 50 pairs of kissing RNA hairpins. Two kissing motifs, 5′CCNY and 5′RYRY, generate highly stable complexes with KDs in the low nanomolar range. Such motifs were introduced in the apical loop of hairpin aptamers that switch between unfolded and folded state upon binding to their cognate target molecule, hence their name aptaswitch. The aptaswitch–ligand complex is specifically recognized by a second RNA hairpin named aptakiss through loop–loop interaction. Taking advantage of our kissing motif repertoire we engineered aptaswitch–aptakiss modules for purine derivatives, namely adenosine, GTP and theophylline and demonstrated that these molecules can be specifically and simultaneously detected by surface plasmon resonance or by fluorescence anisotropy. PMID:27067541

  19. Efficient CRISPR-Mediated Post-Transcriptional Gene Silencing in a Hyperthermophilic Archaeon Using Multiplexed crRNA Expression

    PubMed Central

    Zebec, Ziga; Zink, Isabelle Anna; Kerou, Melina; Schleper, Christa

    2016-01-01

    CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-mediated RNA degradation is catalyzed by a type III system in the hyperthermophilic archaeon Sulfolobus solfataricus. Earlier work demonstrated that the system can be engineered to target specifically mRNA of an endogenous host reporter gene, namely the β-galactosidase in S. solfataricus. Here, we investigated the effect of single and multiple spacers targeting the mRNA of a second reporter gene, α-amylase, at the same, and at different, locations respectively, using a minimal CRISPR (miniCR) locus supplied on a viral shuttle vector. The use of increasing numbers of spacers reduced mRNA levels at progressively higher levels, with three crRNAs (CRISPR RNAs) leading to ∼ 70–80% reduction, and five spacers resulting in an α-amylase gene knockdown of > 90% measured on both mRNA and protein activity levels. Our results indicate that this technology can be used to increase or modulate gene knockdown for efficient post-transcriptional gene silencing in hyperthermophilic archaea, and potentially also in other organisms. PMID:27507792

  20. mCAL: A New Approach for Versatile Multiplex Action of Cas9 Using One sgRNA and Loci Flanked by a Programmed Target Sequence.

    PubMed

    Finnigan, Gregory C; Thorner, Jeremy

    2016-01-01

    Genome editing exploiting CRISPR/Cas9 has been adopted widely in academia and in the biotechnology industry to manipulate DNA sequences in diverse organisms. Molecular engineering of Cas9 itself and its guide RNA, and the strategies for using them, have increased efficiency, optimized specificity, reduced inappropriate off-target effects, and introduced modifications for performing other functions (transcriptional regulation, high-resolution imaging, protein recruitment, and high-throughput screening). Moreover, Cas9 has the ability to multiplex, i.e., to act at different genomic targets within the same nucleus. Currently, however, introducing concurrent changes at multiple loci involves: (i) identification of appropriate genomic sites, especially the availability of suitable PAM sequences; (ii) the design, construction, and expression of multiple sgRNA directed against those sites; (iii) potential difficulties in altering essential genes; and (iv) lingering concerns about "off-target" effects. We have devised a new approach that circumvents these drawbacks, as we demonstrate here using the yeast Saccharomyces cerevisiae First, any gene(s) of interest are flanked upstream and downstream with a single unique target sequence that does not normally exist in the genome. Thereafter, expression of one sgRNA and cotransformation with appropriate PCR fragments permits concomitant Cas9-mediated alteration of multiple genes (both essential and nonessential). The system we developed also allows for maintenance of the integrated, inducible Cas9-expression cassette or its simultaneous scarless excision. Our scheme-dubbed mCAL for " M: ultiplexing of C: as9 at A: rtificial L: oci"-can be applied to any organism in which the CRISPR/Cas9 methodology is currently being utilized. In principle, it can be applied to install synthetic sequences into the genome, to generate genomic libraries, and to program strains or cell lines so that they can be conveniently (and repeatedly

  1. Plasmonic nanohalo optical probes for highly sensitive imaging of survivin mRNA in living cells.

    PubMed

    Qian, Guang-Sheng; Kang, Bin; Zhang, Zhuo-Lei; Li, Xiang-Ling; Zhao, Wei; Xu, Jing-Juan; Chen, Hong-Yuan

    2016-09-25

    A strategy is designed for sensitive detection of tumor biomarker survivin mRNA based on resonance Rayleigh scattering of a single AuNP nanohalo probe that couples large gold nanoparticles (L-AuNPs, 52 nm) with small AuNPs (S-AuNPs, 18 nm) through the affinity interaction between streptavidin and biotin. This core-satellite plasmon ruler is further applied to imaging survivin mRNA in living cells. PMID:27412908

  2. Plasmonic nanohalo optical probes for highly sensitive imaging of survivin mRNA in living cells.

    PubMed

    Qian, Guang-Sheng; Kang, Bin; Zhang, Zhuo-Lei; Li, Xiang-Ling; Zhao, Wei; Xu, Jing-Juan; Chen, Hong-Yuan

    2016-09-25

    A strategy is designed for sensitive detection of tumor biomarker survivin mRNA based on resonance Rayleigh scattering of a single AuNP nanohalo probe that couples large gold nanoparticles (L-AuNPs, 52 nm) with small AuNPs (S-AuNPs, 18 nm) through the affinity interaction between streptavidin and biotin. This core-satellite plasmon ruler is further applied to imaging survivin mRNA in living cells.

  3. Characterizing a novel and sensitive method to measure dsRNA in soil.

    PubMed

    Fischer, Joshua R; Zapata, Fatima; Dubelman, Samuel; Mueller, Geoffrey M; Jensen, Peter D; Levine, Steven L

    2016-10-01

    Performing environmental assessments for double-stranded RNA-based agricultural products require the development of sensitive and selective methods to measure biodegradation rates of dsRNAs. We developed and characterized a novel analytical procedure that uses a molecular hybridization assay (QuantiGene(®)) to accurately measure dsRNA extracted from diverse soils. In this report, we utilize this method to demonstrate that two dsRNAs with distinct size, structure, and sequence degrade rapidly in soil with indistinguishable kinetics.

  4. Sensitive and Selective Detection of HIV-1 RRE RNA Using Vertical Silicon Nanowire Electrode Array

    NASA Astrophysics Data System (ADS)

    Lee, Jaehyung; Hong, Min-Ho; Han, Sanghun; Na, Jukwan; Kim, Ilsoo; Kwon, Yong-Joon; Lim, Yong-beom; Choi, Heon-Jin

    2016-07-01

    In this study, HIV-1 Rev response element (RRE) RNA was detected via an Au-coated vertical silicon nanowire electrode array (VSNEA). The VSNEA was fabricated by combining bottom-up and top-down approaches and then immobilized by artificial peptides for the recognition of HIV-1 RRE. Differential pulse voltammetry (DPV) analysis was used to measure the electrochemical response of the peptide-immobilized VSNEA to the concentration and types of HIV-1 RRE RNA. DPV peaks showed linearity to the concentration of RNA with a detection limit down to 1.513 fM. It also showed the clear different peaks to the mutated HIV-1 RRE RNA. The high sensitivity and selectivity of VSNEA for the detection of HIV-1 RRE RNA may be attributed to the high surface-to-volume ratio and total overlap diffusion mode of ions of the one-dimensional nanowire electrodes.

  5. A focus on microfluidics and nanotechnology approaches for the ultra sensitive detection of microRNA.

    PubMed

    Lingam, Swathi; Beta, Madhu; Dendukuri, Dhananjay; Krishnakumar, Subramanian

    2014-01-01

    MicroRNAs are small RNAs that are deregulated under disease conditions. This allows them to be used as biomarkers for disease diagnosis. Recently, such microRNAs are serving as non- invasive blood based biomarkers due to ease of detection, and high stability at room temperature in biofluids. In view of these advancements in biomarker research, efficient miRNA detection at the femtomolar level is important. MiRNA detection techniques to date include Northern blotting, real time PCR and microarray technology. While these are efficient techniques, they cannot detect very low miRNA levels, and have questionable specificity. This review describes nanotechnology, microfluidics and liquid phase miRNA detection techniques that are highly sensitive and specific. With some sophistication, some of these techniques can be used as point of care devices to rapidly detect low level miRNA in patient clinical samples to aid disease diagnosis and prognosis.

  6. Sensitive and Selective Detection of HIV-1 RRE RNA Using Vertical Silicon Nanowire Electrode Array.

    PubMed

    Lee, Jaehyung; Hong, Min-Ho; Han, Sanghun; Na, Jukwan; Kim, Ilsoo; Kwon, Yong-Joon; Lim, Yong-Beom; Choi, Heon-Jin

    2016-12-01

    In this study, HIV-1 Rev response element (RRE) RNA was detected via an Au-coated vertical silicon nanowire electrode array (VSNEA). The VSNEA was fabricated by combining bottom-up and top-down approaches and then immobilized by artificial peptides for the recognition of HIV-1 RRE. Differential pulse voltammetry (DPV) analysis was used to measure the electrochemical response of the peptide-immobilized VSNEA to the concentration and types of HIV-1 RRE RNA. DPV peaks showed linearity to the concentration of RNA with a detection limit down to 1.513 fM. It also showed the clear different peaks to the mutated HIV-1 RRE RNA. The high sensitivity and selectivity of VSNEA for the detection of HIV-1 RRE RNA may be attributed to the high surface-to-volume ratio and total overlap diffusion mode of ions of the one-dimensional nanowire electrodes.

  7. Label-Free Isothermal Amplification Assay for Specific and Highly Sensitive Colorimetric miRNA Detection

    PubMed Central

    2016-01-01

    We describe a new method for the detection of miRNA in biological samples. This technology is based on the isothermal nicking enzyme amplification reaction and subsequent hybridization of the amplification product with gold nanoparticles and magnetic microparticles (barcode system) to achieve naked-eye colorimetric detection. This platform was used to detect a specific miRNA (miRNA-10b) associated with breast cancer, and attomolar sensitivity was demonstrated. The assay was validated in cell culture lysates from breast cancer cells and in serum from a mouse model of breast cancer. PMID:27713932

  8. Ratiometric bimolecular beacons for the sensitive detection of RNA in single living cells.

    PubMed

    Chen, Antony K; Davydenko, Olga; Behlke, Mark A; Tsourkas, Andrew

    2010-08-01

    Numerous studies have utilized molecular beacons (MBs) to image RNA expression in living cells; however, there is growing evidence that the sensitivity of RNA detection is significantly hampered by their propensity to emit false-positive signals. To overcome these limitations, we have developed a new RNA imaging probe called ratiometric bimolecular beacon (RBMB), which combines functional elements of both conventional MBs and siRNA. Analogous to MBs, RBMBs elicit a fluorescent reporter signal upon hybridization to complementary RNA. In addition, an siRNA-like double-stranded domain is used to facilitate nuclear export. Accordingly, live-cell fluorescent imaging showed that RBMBs are localized predominantly in the cytoplasm, whereas MBs are sequestered into the nucleus. The retention of RBMBs within the cytoplasmic compartment led to >15-fold reduction in false-positive signals and a significantly higher signal-to-background compared with MBs. The RBMBs were also designed to possess an optically distinct reference fluorophore that remains unquenched regardless of probe confirmation. This reference dye not only provided a means to track RBMB localization, but also allowed single cell measurements of RBMB fluorescence to be corrected for variations in probe delivery. Combined, these attributes enabled RBMBs to exhibit an improved sensitivity for RNA detection in living cells.

  9. Highly sensitive and ultrafast read mapping for RNA-seq analysis.

    PubMed

    Medina, I; Tárraga, J; Martínez, H; Barrachina, S; Castillo, M I; Paschall, J; Salavert-Torres, J; Blanquer-Espert, I; Hernández-García, V; Quintana-Ortí, E S; Dopazo, J

    2016-04-01

    As sequencing technologies progress, the amount of data produced grows exponentially, shifting the bottleneck of discovery towards the data analysis phase. In particular, currently available mapping solutions for RNA-seq leave room for improvement in terms of sensitivity and performance, hindering an efficient analysis of transcriptomes by massive sequencing. Here, we present an innovative approach that combines re-engineering, optimization and parallelization. This solution results in a significant increase of mapping sensitivity over a wide range of read lengths and substantial shorter runtimes when compared with current RNA-seq mapping methods available. PMID:26740642

  10. Facile semi-automated forensic body fluid identification by multiplex solution hybridization of NanoString® barcode probes to specific mRNA targets.

    PubMed

    Danaher, Patrick; White, Robin Lynn; Hanson, Erin K; Ballantyne, Jack

    2015-01-01

    A DNA profile from the perpetrator does not reveal, per se, the circumstances by which it was transferred. Body fluid identification by mRNA profiling may allow extraction of contextual 'activity level' information from forensic samples. Here we describe the development of a prototype multiplex digital gene expression (DGE) method for forensic body fluid/tissue identification based upon solution hybridization of color-coded NanoString(®) probes to 23 mRNA targets. The method identifies peripheral blood, semen, saliva, vaginal secretions, menstrual blood and skin. We showed that a simple 5 min room temperature cellular lysis protocol gave equivalent results to standard RNA isolation from the same source material, greatly enhancing the ease-of-use of this method in forensic sample processing. We first describe a model for gene expression in a sample from a single body fluid and then extend that model to mixtures of body fluids. We then describe calculation of maximum likelihood estimates (MLEs) of body fluid quantities in a sample, and we describe the use of likelihood ratios to test for the presence of each body fluid in a sample. Known single source samples of blood, semen, vaginal secretions, menstrual blood and skin all demonstrated the expected tissue-specific gene expression for at least two of the chosen biomarkers. Saliva samples were more problematic, with their previously identified characteristic genes exhibiting poor specificity. Nonetheless the most specific saliva biomarker, HTN3, was expressed at a higher level in saliva than in any of the other tissues. Crucially, our algorithm produced zero false positives across this study's 89 unique samples. As a preliminary indication of the ability of the method to discern admixtures of body fluids, five mixtures were prepared. The identities of the component fluids were evident from the gene expression profiles of four of the five mixtures. Further optimization of the biomarker 'CodeSet' will be required

  11. Stem-loop DNA-assisted silicon nanowires-based biochemical sensors with ultra-high sensitivity, specificity, and multiplexing capability.

    PubMed

    Xie, Juan; Jiang, Xiangxu; Zhong, Yiling; Lu, Yimei; Wang, Siyi; Wei, Xinpan; Su, Yuanyuan; He, Yao

    2014-08-01

    A class of stem-loop DNA-assisted silicon nanowires (SiNWs)-based fluorescent biosensor is presented in this report. Significantly, the sensor enables rapid and sensitive detection of DNA targets with a concentration as low as 1 pM. Moreover, the large planar surface of SiNWs facilitates simultaneous assembly with different DNA strands, which is favorable for multiplexed DNA detection. On the other hand, the SiNWs-based sensor is highly efficacious for detecting heavy metal ions. Mercury ions (Hg(2+)) of low concentrations (e.g., 5 pM) are readily identified from its mixture with over 10 kinds of interfering metal ions, even in real water samples. Given that SiNWs can be fabricated in a facile, reproducible and low-cost manner, this kind of SiNWs-based high-performance sensor is expected to be a practical analytical tool for a variety of biological and environment-protection applications.

  12. Unusual diversity in alpha-amanitin sensitivity of RNA polymerases in trichomonads.

    PubMed

    Vanácová, S; Tachezy, J; Ullu, E; Tschudi, C

    2001-07-01

    Previous studies in the parasitic protist Trichomonas vaginalis have revealed that protein coding genes are transcribed by an alpha-amanitin-resistant RNA polymerase (RNAP) II. To investigate whether this unusual property is a general characteristic of trichomonads, we addressed the physiology of RNA synthesis in lysolecithin-permeabilized cells. Unlike in T. vaginalis, RNAP II in Tritrichomonas foetus was highly sensitive to the inhibitor alpha-amanitin. On the other hand, RNAP III, identified by its sensitivity to the specific inhibitor tagetitoxin, was found to be resistant to alpha-amanitin in Tritrichomonas foetus, but showed a typical intermediate sensitivity in T. vaginalis. Extension of this study to an additional seven trichomonad species confirmed this genera specific pattern of alpha-amanitin sensitivity and highlighted an unusual diversity in RNAPs among trichomonads, a closely related group of unicellular eukaryotes. PMID:11420110

  13. MicroRNA-221 and -222 Regulate Radiation Sensitivity by Targeting the PTEN Pathway

    SciTech Connect

    Zhang Chunzhi; Kang Chunsheng; Wang Ping; Cao Yongzhen; Lv Zhonghong; Yu Shizhu; Wang Guangxiu; Zhang Anling; Jia Zhifan; Han Lei; Yang Chunying; Ishiyama, Hiromichi; Teh, Bin S.; Xu Bo; Pu Peiyu

    2011-05-01

    Purpose: MicroRNAs (miRNAs) are noncoding RNAs inhibiting expression of numerous target genes by posttranscriptional regulation. miRNA-221 and miRNA-222 (miRNA-221/-222) expression is elevated in radioresistant tumor cell lines; however, it is not known whether and how miRNAs control cellular responses to ionizing irradiation. Methods and Materials: We used bioinformatic analyses, luciferase reporter assay, and genetic knockdown and biochemical assays to characterize the regulation pathways of miRNA-221/-222 in response to radiation treatment. Results: We identified the PTEN gene as a target of miRNA-221/-222. Furthermore, we found that knocking down miRNA-221/-222 by antisense oligonucleotides upregulated PTEN expression. Upregulated PTEN expression suppressed AKT activity and increased radiation-induced apoptosis, resulting in enhancement of radiosensitivity in tumor cells. Conclusions: miRNA-221/-222 control radiation sensitivity by regulating the PTEN/AKT pathway and can be explored as novel targets for radiosensitization.

  14. Multiplexed digital quantification of binge-like alcohol-mediated alterations in maternal uterine angiogenic mRNA transcriptome.

    PubMed

    Ramadoss, Jayanth; Magness, Ronald R

    2012-06-01

    Genomic studies on fetal alcohol spectrum disorders (FASD) have utilized either genome-wide microarrays/bioinformatics or targeted real-time PCR (RT-PCR). We utilized herein for the first time a novel digital approach with high throughput as well as the capability to focus on one physiological system. The aim of the present study was to investigate alcohol-induced alterations in uterine angiogenesis-related mRNA abundance using digital mRNA technology. Four biological and three technical replicates of uterine arterial endothelial cells from third-trimester ewes were fluorescence-activated cell sorted, validated, and treated without or with binge-like alcohol. A capture probe covalently bound to an oligonucleotide containing biotin and a color-coded reporter probe were designed for 85 angiogenesis-related genes and analyzed with the Nanostring nCounter system. Twenty genes were downregulated (↓) and two upregulated (↑), including angiogenic growth factors/receptors (↓placental growth factor), adhesion molecules (↓angiopoietin-like-3; ↓collagen-18A1; ↓endoglin), proteases/matrix proteins/inhibitors (↓alanyl aminopeptidase; ↓collagen-4A3; ↓heparanase; ↓plasminogen, ↑plasminogen activator urokinase; ↓platelet factor-4; ↓plexin domain containing-1; ↓tissue inhibitor of metalloproteinases-3), transcription/signaling molecules (↓heart and neural crest derivatives-2; ↓DNA-binding protein inhibitor; ↓NOTCH-4; ↓ribosomal protein-L13a1; ↓ribosomal protein large-P1), cytokines/chemokines (↓interleukin-1B), and miscellaneous growth factors (↓leptin; ↓platelet-derived growth factor-α); ↓transforming growth factor (TGF-α; ↑TGF-β receptor-1). These novel data show significant detrimental alcohol effects on genes controlling angiogenesis supporting a mechanistic role for abnormal uteroplacental vascular development in FASD. The tripartite digital gene expression system is therefore a valuable tool to answer many additional

  15. A rapid and sensitive method to detect siRNA-mediated mRNA cleavage in vivo using 5' RACE and a molecular beacon probe.

    PubMed

    Lasham, Annette; Herbert, Mike; Coppieters 't Wallant, Natacha; Patel, Rachna; Feng, Sheryl; Eszes, Marika; Cao, Helen; Reid, Glen

    2010-01-01

    Specific detection of mRNA cleavage by 5'RACE is the only method to confirm the knockdown of mRNA by RNA interference, but is rarely reported for in vivo studies. We have combined 5'-RNA-linker-mediated RACE (5'-RLM-RACE) with real-time PCR using a molecular beacon to develop a rapid and specific method termed MBRACE, which we have used to detect small-interfering RNA (siRNA)-induced cleavage of ApoB, RRM1 and YBX1 transcripts in vitro, and ApoB in vivo. When RNA from siRNA-transfected cells was used for 5'-RLM-RACE and a cleavage site-specific molecular beacon probe was included in subsequent real-time PCR analysis, the specific mRNA cleavage product was detected. Detection of siRNA-mediated cleavage was also observed when RNA from mouse liver following administration of ApoB-specific siRNA was analysed, even in cases where ApoB knockdown measured by real-time PCR was <10%. With its sensitivity and specificity, this variation on the 5'RACE method should prove a useful tool to detect mRNA cleavage and corroborate knockdown studies following siRNA use in vivo.

  16. GABAergic mRNA expression is upregulated in the prefrontal cortex of rats sensitized to methamphetamine.

    PubMed

    Wearne, Travis A; Parker, Lindsay M; Franklin, Jane L; Goodchild, Ann K; Cornish, Jennifer L

    2016-01-15

    Inhibitory gamma-aminobutyric acid (GABA)-mediated neurotransmission plays an important role in the regulation of the prefrontal cortex (PFC), with increasing evidence suggesting that dysfunctional GABAergic processing of the PFC may underlie certain deficits reported across psychotic disorders. Methamphetamine (METH) is a psychostimulant that induces chronic psychosis in a subset of users, with repeat administration producing a progressively increased vulnerability to psychotic relapse following subsequent drug administration (sensitization). The aim here was to investigate changes to GABAergic mRNA expression in the PFC of rats sensitized to METH using quantitative polymerase chain reaction (qPCR). Male Sprague-Dawley rats (n=12) underwent repeated methamphetamine (intraperitoneal (i.p.) or saline injections for 7 days. Following 14 days of withdrawal, rats were challenged with acute methamphetamine (1mg/kg i.p.) and RNA was isolated from the PFC to compare the relative mRNA expression of a range of GABA enzymes, transporters and receptors subunits. METH challenge resulted in a significant sensitized behavioral (locomotor) response in METH pre-treated animals compared with saline pre-treated controls. The mRNAs of transporters (GAT1 and GAT3), ionotropic GABAA receptor subunits (α3 and β1), together with the metabotropic GABAB1 receptor, were upregulated in the PFC of sensitized rats compared with saline controls. These findings indicate that GABAergic mRNA expression is significantly altered at the pre and postsynaptic level following sensitization to METH, with sensitization resulting in the transcriptional upregulation of several inhibitory genes. These changes likely have significant consequences on GABA-mediated neurotransmission in the PFC and may underlie certain symptoms conserved across psychotic disorders, such as executive dysfunction.

  17. A handheld flow genetic analysis system (FGAS): towards rapid, sensitive, quantitative and multiplex molecular diagnosis at the point-of-care level.

    PubMed

    Shu, Bowen; Zhang, Chunsun; Xing, Da

    2015-06-21

    A handheld flow genetic analysis system (FGAS) is proposed for rapid, sensitive, multiplex and real-time quantification of nucleic acids at the point-of-care (POC) level. The FGAS includes a helical thermal-gradient microreactor and a microflow actuator, as well as control circuitry for temperature, fluid and power management, and smartphone fluorescence imaging. All of these features are integrated into a field-portable and easy-to-use molecular diagnostic platform powered by lithium batteries. Due to the unique design of the microreactor, not only steady temperatures for denaturation and annealing/extension but also a linear thermal gradient for spatial high-resolution melting can be achieved through simply maintaining a single heater at constant temperature. The smartphone fluorescence imaging system has a wide field of view that captures all PCR channels of the microreactor in a single snapshot without the need for any mechanical scanning. By these designs, the FGAS enables real-time monitoring of the temporal and spatial fluorescence signatures of amplicons during continuous-flow amplification. On the current FGAS, visual detection of as little as 10 copies per μL of genomic DNA of Salmonella enterica was achieved in 15 min, with real-time quantitative detection of the DNA over 6 orders of magnitude concentration from 10(6) to 10(1) copies per μL also completed in 7.5-15 min. In addition, multiple pathogenic DNA targets could be simultaneously discriminated with direct bar-chart readout or multiplex spatial melting in serial flow. We anticipate that the FGAS has great potential to become a next-generation gene analyzer for POC molecular diagnostics. PMID:25953325

  18. 3'READS+, a sensitive and accurate method for 3' end sequencing of polyadenylated RNA.

    PubMed

    Zheng, Dinghai; Liu, Xiaochuan; Tian, Bin

    2016-10-01

    Sequencing of the 3' end of poly(A)(+) RNA identifies cleavage and polyadenylation sites (pAs) and measures transcript expression. We previously developed a method, 3' region extraction and deep sequencing (3'READS), to address mispriming issues that often plague 3' end sequencing. Here we report a new version, named 3'READS+, which has vastly improved accuracy and sensitivity. Using a special locked nucleic acid oligo to capture poly(A)(+) RNA and to remove the bulk of the poly(A) tail, 3'READS+ generates RNA fragments with an optimal number of terminal A's that balance data quality and detection of genuine pAs. With improved RNA ligation steps for efficiency, the method shows much higher sensitivity (over two orders of magnitude) compared to the previous version. Using 3'READS+, we have uncovered a sizable fraction of previously overlooked pAs located next to or within a stretch of adenylate residues in human genes and more accurately assessed the frequency of alternative cleavage and polyadenylation (APA) in HeLa cells (∼50%). 3'READS+ will be a useful tool to accurately study APA and to analyze gene expression by 3' end counting, especially when the amount of input total RNA is limited. PMID:27512124

  19. 3'READS+, a sensitive and accurate method for 3' end sequencing of polyadenylated RNA.

    PubMed

    Zheng, Dinghai; Liu, Xiaochuan; Tian, Bin

    2016-10-01

    Sequencing of the 3' end of poly(A)(+) RNA identifies cleavage and polyadenylation sites (pAs) and measures transcript expression. We previously developed a method, 3' region extraction and deep sequencing (3'READS), to address mispriming issues that often plague 3' end sequencing. Here we report a new version, named 3'READS+, which has vastly improved accuracy and sensitivity. Using a special locked nucleic acid oligo to capture poly(A)(+) RNA and to remove the bulk of the poly(A) tail, 3'READS+ generates RNA fragments with an optimal number of terminal A's that balance data quality and detection of genuine pAs. With improved RNA ligation steps for efficiency, the method shows much higher sensitivity (over two orders of magnitude) compared to the previous version. Using 3'READS+, we have uncovered a sizable fraction of previously overlooked pAs located next to or within a stretch of adenylate residues in human genes and more accurately assessed the frequency of alternative cleavage and polyadenylation (APA) in HeLa cells (∼50%). 3'READS+ will be a useful tool to accurately study APA and to analyze gene expression by 3' end counting, especially when the amount of input total RNA is limited.

  20. Fast Hepatitis C Virus RNA Elimination and NS5A Redistribution by NS5A Inhibitors Studied by a Multiplex Assay Approach

    PubMed Central

    Liu, Dandan; Ji, Juan; Ndongwe, Tanya P.; Michailidis, Eleftherios; Rice, Charles M.; Ralston, Robert

    2015-01-01

    While earlier therapeutic strategies for the treatment of hepatitis C virus (HCV) infection relied exclusively on interferon (IFN) and ribavirin (RBV), four direct-acting antiviral agents (DAAs) have now been approved, aiming for an interferon-free strategy with a short treatment duration and fewer side effects. To facilitate studies on the mechanism of action (MOA) and efficacy of DAAs, we established a multiplex assay approach, which employs flow cytometry, a Gaussia luciferase reporter system, Western blot analysis, reverse transcription-quantitative PCR (RT-qPCR), a limited dilution assay (50% tissue culture infectious dose [TCID50]), and an image profiling assay that follows the NS5A redistribution in response to drug treatment. We used this approach to compare the relative potency of various DAAs and the kinetics of their antiviral effects as a potential preclinical measure of their potential clinical utility. We evaluated the NS5A inhibitors ledipasvir (LDV) and daclatasvir (DCV), the NS3/4A inhibitor danoprevir (DNV), and the NS5B inhibitor sofosbuvir (SOF). In terms of kinetics, our data demonstrate that the NS5A inhibitor LDV, followed closely by DCV, has the fastest effect on suppression of viral proteins and RNA and on redistribution of NS5A. In terms of MOA, LDV has a more pronounced effect than DCV on the viral replication, assembly, and infectivity of released virus. Our approach can be used to facilitate the study of the biological processes involved in HCV replication and help identify optimal drug combinations. PMID:25845863

  1. Fast hepatitis C virus RNA elimination and NS5A redistribution by NS5A inhibitors studied by a multiplex assay approach.

    PubMed

    Liu, Dandan; Ji, Juan; Ndongwe, Tanya P; Michailidis, Eleftherios; Rice, Charles M; Ralston, Robert; Sarafianos, Stefan G

    2015-01-01

    While earlier therapeutic strategies for the treatment of hepatitis C virus (HCV) infection relied exclusively on interferon (IFN) and ribavirin (RBV), four direct-acting antiviral agents (DAAs) have now been approved, aiming for an interferon-free strategy with a short treatment duration and fewer side effects. To facilitate studies on the mechanism of action (MOA) and efficacy of DAAs, we established a multiplex assay approach, which employs flow cytometry, a Gaussia luciferase reporter system, Western blot analysis, reverse transcription-quantitative PCR (RT-qPCR), a limited dilution assay (50% tissue culture infectious dose [TCID50]), and an image profiling assay that follows the NS5A redistribution in response to drug treatment. We used this approach to compare the relative potency of various DAAs and the kinetics of their antiviral effects as a potential preclinical measure of their potential clinical utility. We evaluated the NS5A inhibitors ledipasvir (LDV) and daclatasvir (DCV), the NS3/4A inhibitor danoprevir (DNV), and the NS5B inhibitor sofosbuvir (SOF). In terms of kinetics, our data demonstrate that the NS5A inhibitor LDV, followed closely by DCV, has the fastest effect on suppression of viral proteins and RNA and on redistribution of NS5A. In terms of MOA, LDV has a more pronounced effect than DCV on the viral replication, assembly, and infectivity of released virus. Our approach can be used to facilitate the study of the biological processes involved in HCV replication and help identify optimal drug combinations. PMID:25845863

  2. Characterizing a novel and sensitive method to measure dsRNA in soil.

    PubMed

    Fischer, Joshua R; Zapata, Fatima; Dubelman, Samuel; Mueller, Geoffrey M; Jensen, Peter D; Levine, Steven L

    2016-10-01

    Performing environmental assessments for double-stranded RNA-based agricultural products require the development of sensitive and selective methods to measure biodegradation rates of dsRNAs. We developed and characterized a novel analytical procedure that uses a molecular hybridization assay (QuantiGene(®)) to accurately measure dsRNA extracted from diverse soils. In this report, we utilize this method to demonstrate that two dsRNAs with distinct size, structure, and sequence degrade rapidly in soil with indistinguishable kinetics. PMID:27441991

  3. A Genome-Wide siRNA Screen to Identify Modulators of Insulin Sensitivity and Gluconeogenesis

    PubMed Central

    Yang, Ruojing; Lacson, Raul G.; Castriota, Gino; Zhang, Xiaohua D.; Liu, Yaping; Zhao, Wenqing; Einstein, Monica; Camargo, Luiz Miguel; Qureshi, Sajjad; Wong, Kenny K.; Zhang, Bei B.; Ferrer, Marc; Berger, Joel P.

    2012-01-01

    Background Hepatic insulin resistance impairs insulin’s ability to suppress hepatic glucose production (HGP) and contributes to the development of type 2 diabetes (T2D). Although the interests to discover novel genes that modulate insulin sensitivity and HGP are high, it remains challenging to have a human cell based system to identify novel genes. Methodology/Principal Findings To identify genes that modulate hepatic insulin signaling and HGP, we generated a human cell line stably expressing beta-lactamase under the control of the human glucose-6-phosphatase (G6PC) promoter (AH-G6PC cells). Both beta-lactamase activity and endogenous G6PC mRNA were increased in AH-G6PC cells by a combination of dexamethasone and pCPT-cAMP, and reduced by insulin. A 4-gene High-Throughput-Genomics assay was developed to concomitantly measure G6PC and pyruvate-dehydrogenase-kinase-4 (PDK4) mRNA levels. Using this assay, we screened an siRNA library containing pooled siRNA targeting 6650 druggable genes and identified 614 hits that lowered G6PC expression without increasing PDK4 mRNA levels. Pathway analysis indicated that siRNA-mediated knockdown (KD) of genes known to positively or negatively affect insulin signaling increased or decreased G6PC mRNA expression, respectively, thus validating our screening platform. A subset of 270 primary screen hits was selected and 149 hits were confirmed by target gene KD by pooled siRNA and 7 single siRNA for each gene to reduce G6PC expression in 4-gene HTG assay. Subsequently, pooled siRNA KD of 113 genes decreased PEPCK and/or PGC1alpha mRNA expression thereby demonstrating their role in regulating key gluconeogenic genes in addition to G6PC. Last, KD of 61 of the above 113 genes potentiated insulin-stimulated Akt phosphorylation, suggesting that they suppress gluconeogenic gene by enhancing insulin signaling. Conclusions/Significance These results support the proposition that the proteins encoded by the genes identified in our cell

  4. Simple and sensitive fluorescence detection of the RNA endonuclease activity of mammalian argonaute2 protein based on an RNA molecular beacon.

    PubMed

    Li, Feng; Li, Peng; Yang, Limin; Tang, Bo

    2012-12-28

    A new strategy for determining the RNA endonuclease activity of mammalian argonaute2 (Ago2) protein has been developed, which combines the unique cleavage function of Ago2 protein with an RNA molecular beacon (RMB). Through the fluorescence restoration of the RMB, simple and sensitive detection of Ago2 is achieved.

  5. Stem-loop DNA-assisted silicon nanowires-based biochemical sensors with ultra-high sensitivity, specificity, and multiplexing capability

    NASA Astrophysics Data System (ADS)

    Xie, Juan; Jiang, Xiangxu; Zhong, Yiling; Lu, Yimei; Wang, Siyi; Wei, Xinpan; Su, Yuanyuan; He, Yao

    2014-07-01

    A class of stem-loop DNA-assisted silicon nanowires (SiNWs)-based fluorescent biosensor is presented in this report. Significantly, the sensor enables rapid and sensitive detection of DNA targets with a concentration as low as 1 pM. Moreover, the large planar surface of SiNWs facilitates simultaneous assembly with different DNA strands, which is favorable for multiplexed DNA detection. On the other hand, the SiNWs-based sensor is highly efficacious for detecting heavy metal ions. Mercury ions (Hg2+) of low concentrations (e.g., 5 pM) are readily identified from its mixture with over 10 kinds of interfering metal ions, even in real water samples. Given that SiNWs can be fabricated in a facile, reproducible and low-cost manner, this kind of SiNWs-based high-performance sensor is expected to be a practical analytical tool for a variety of biological and environment-protection applications.A class of stem-loop DNA-assisted silicon nanowires (SiNWs)-based fluorescent biosensor is presented in this report. Significantly, the sensor enables rapid and sensitive detection of DNA targets with a concentration as low as 1 pM. Moreover, the large planar surface of SiNWs facilitates simultaneous assembly with different DNA strands, which is favorable for multiplexed DNA detection. On the other hand, the SiNWs-based sensor is highly efficacious for detecting heavy metal ions. Mercury ions (Hg2+) of low concentrations (e.g., 5 pM) are readily identified from its mixture with over 10 kinds of interfering metal ions, even in real water samples. Given that SiNWs can be fabricated in a facile, reproducible and low-cost manner, this kind of SiNWs-based high-performance sensor is expected to be a practical analytical tool for a variety of biological and environment-protection applications. Electronic supplementary information (ESI) available. See DOI: 10.1039/c4nr01097c

  6. Molecular structure and thermodynamic predictions to create highly sensitive microRNA biosensors.

    PubMed

    Larkey, Nicholas E; Brucks, Corinne N; Lansing, Shan S; Le, Sophia D; Smith, Natasha M; Tran, Victoria; Zhang, Lulu; Burrows, Sean M

    2016-02-25

    Many studies have established microRNAs (miRNAs) as post-transcriptional regulators in a variety of intracellular molecular processes. Abnormal changes in miRNA have been associated with several diseases. However, these changes are sometimes subtle and occur at nanomolar levels or lower. Several biosensing hurdles for in situ cellular/tissue analysis of miRNA limit detection of small amounts of miRNA. Of these limitations the most challenging are selectivity and sensor degradation creating high background signals and false signals. Recently we developed a reporter+probe biosensor for let-7a that showed potential to mitigate false signal from sensor degradation. Here we designed reporter+probe biosensors for miR-26a-2-3p and miR-27a-5p to better understand the effect of thermodynamics and molecular structures of the biosensor constituents on the analytical performance. Signal changes from interactions between Cy3 and Cy5 on the reporters were used to understand structural aspects of the reporter designs. Theoretical thermodynamic values, single stranded conformations, hetero- and homodimerization structures, and equilibrium concentrations of the reporters and probes were used to interpret the experimental observations. Studies of the sensitivity and selectivity revealed 5-9 nM detection limits in the presence and absence of interfering off-analyte miRNAs. These studies will aid in determining how to rationally design reporter+probe biosensors to overcome hurdles associated with highly sensitive miRNA biosensing.

  7. Influenza A virus drift variants reduced the detection sensitivity of a commercial multiplex nucleic acid amplification assay in the season 2014/15.

    PubMed

    Huzly, Daniela; Korn, Klaus; Bierbaum, Sibylle; Eberle, Björn; Falcone, Valeria; Knöll, Antje; Steininger, Philipp; Panning, Marcus

    2016-09-01

    The influenza season 2014/15 was dominated by drift variants of influenza A(H3N2), which resulted in a reduced vaccine effectiveness. It was not clear if the performance of commercial nucleic-acid-based amplification (NAT) assays for the detection of influenza was affected. The purpose of this study was to perform a real-life evaluation of two commercial NAT assays. During January-April 2015, we tested a total of 665 samples from patients with influenza-like illness using the Fast Track Diagnostics Respiratory pathogens 21, a commercial multiplex kit, (cohorts 1 and 2, n = 563 patients) and the Xpert Flu/RSV XC assay (cohort 3, n = 102 patients), a single-use cartridge system. An in-house influenza real-time RT-PCR (cohort 1) and the RealStar Influenza RT-PCR 1.0 Kit (cohort 2 and 3) served as reference tests. Compared to the reference assay, an overall agreement of 95.9 % (cohort 1), 95 % (cohort 2), and 98 % (cohort 3) was achieved. A total of 24 false-negative results were observed using the Fast Track Diagnostics Respiratory pathogens 21 kit. No false-negative results occurred using the Xpert Flu/RSV XC assay. The Fast Track Diagnostics Respiratory pathogens 21 kit and the Xpert Flu/RSV XC assay had sensitivities of 90.7 % and 100 % and specificities of 100 % and 94.1 %, respectively, compared to the RealStar 1.0 kit. Upon modification of the Fast Track Diagnostics Respiratory pathogens 21 kit, the sensitivity increased to 97.3 %. Influenza virus strains circulating during the 2014/15 season reduced the detection sensitivity of a commercial NAT assay, and continuous monitoring of test performance is therefore necessary. PMID:27316440

  8. Cold-Sensitive Pseudomonas RNA Polymerase II. Cold-Promoted Restriction of Bacteriophage CB3 and the Lack of Host-Dependent Bacteriophage-Specific RNA Transcription

    PubMed Central

    Sobieski, Rodney J.; Olsen, Ronald H.

    1973-01-01

    Cold-sensitive restriction of Pseudomonas phage CB3 by Pseudomonas aeruginosa strain PAT2 involves some aspect of CB3 specific RNA synthesis at 20 C. Experiments using chloramphenicol treatment and RNA-DNA hybridization establish that the amount of CB3 RNA present at 20 C is consistent with the known percentage of phage yielder cells at 20 C. Thus, it appears that nonyielder cells of PAT2 synthesize little or no phage-specific mRNA. Burgess technique extracted PAT2 RNA polymerase (RNAP) is cold sensitive when assayed in vitro with CB3 DNA at 20 C. However, it is not cold sensitive when either calf thymus or PAT2 DNA are the templates for transcription. Low ionic strength assay conditions eliminate the cold sensitivity of PAT2 RNAP. The effect of low ionic environments on transcription initiation along with the in vivo and in vitro suppression of cold sensitivity by host rifampin resistance suggests that the inability of CB3 to reproduce in PAT2 at 20 C is a cold-sensitive step in host RNAP initiation. Our modified RNAP extraction procedure for PAT2 and PAO1C also results in the recovery of cold-sensitive PAT2 RNAP with respect to CB3 DNA templates and points to basic enzymological differences between the two hosts. A model is presented for the unusual influence of temperature on the initiation process of both PAT2 and PAO1C on RNAP transcription. PMID:4202618

  9. Multiplex, Rapid, and Sensitive Isothermal Detection of Nucleic-Acid Sequence by Endonuclease Restriction-Mediated Real-Time Multiple Cross Displacement Amplification.

    PubMed

    Wang, Yi; Wang, Yan; Zhang, Lu; Liu, Dongxin; Luo, Lijuan; Li, Hua; Cao, Xiaolong; Liu, Kai; Xu, Jianguo; Ye, Changyun

    2016-01-01

    We have devised a novel isothermal amplification technology, termed endonuclease restriction-mediated real-time multiple cross displacement amplification (ET-MCDA), which facilitated multiplex, rapid, specific and sensitive detection of nucleic-acid sequences at a constant temperature. The ET-MCDA integrated multiple cross displacement amplification strategy, restriction endonuclease cleavage and real-time fluorescence detection technique. In the ET-MCDA system, the functional cross primer E-CP1 or E-CP2 was constructed by adding a short sequence at the 5' end of CP1 or CP2, respectively, and the new E-CP1 or E-CP2 primer was labeled at the 5' end with a fluorophore and in the middle with a dark quencher. The restriction endonuclease Nb.BsrDI specifically recognized the short sequence and digested the newly synthesized double-stranded terminal sequences (5' end short sequences and their complementary sequences), which released the quenching, resulting on a gain of fluorescence signal. Thus, the ET-MCDA allowed real-time detection of single or multiple targets in only a single reaction, and the positive results were observed in as short as 12 min, detecting down to 3.125 fg of genomic DNA per tube. Moreover, the analytical specificity and the practical application of the ET-MCDA were also successfully evaluated in this study. Here, we provided the details on the novel ET-MCDA technique and expounded the basic ET-MCDA amplification mechanism. PMID:27242766

  10. Stem-loop DNA-assisted silicon nanowires-based biochemical sensors with ultra-high sensitivity, specificity, and multiplexing capability.

    PubMed

    Xie, Juan; Jiang, Xiangxu; Zhong, Yiling; Lu, Yimei; Wang, Siyi; Wei, Xinpan; Su, Yuanyuan; He, Yao

    2014-08-01

    A class of stem-loop DNA-assisted silicon nanowires (SiNWs)-based fluorescent biosensor is presented in this report. Significantly, the sensor enables rapid and sensitive detection of DNA targets with a concentration as low as 1 pM. Moreover, the large planar surface of SiNWs facilitates simultaneous assembly with different DNA strands, which is favorable for multiplexed DNA detection. On the other hand, the SiNWs-based sensor is highly efficacious for detecting heavy metal ions. Mercury ions (Hg(2+)) of low concentrations (e.g., 5 pM) are readily identified from its mixture with over 10 kinds of interfering metal ions, even in real water samples. Given that SiNWs can be fabricated in a facile, reproducible and low-cost manner, this kind of SiNWs-based high-performance sensor is expected to be a practical analytical tool for a variety of biological and environment-protection applications. PMID:24981573

  11. Silicon on-chip side-coupled high-Q micro-cavities for the multiplexing of high sensitivity photonic crystal integrated sensors array

    NASA Astrophysics Data System (ADS)

    Yang, Daquan; Wang, Chunhong; Yuan, Wei; Wang, Bo; Yang, Yujie; Ji, Yuefeng

    2016-09-01

    A novel two-dimensional (2D) silicon (Si) photonic crystal (PC) α-H0-slot micro-cavity with high Q-factor and high sensitivity (S) is presented. Based on the proposed α-H0-Slot micro-cavities, an optimal design of photonic crystal integrated sensors array (PC-ISA) on monolithic silicon on insulator (SOI) is displayed. By using finite-difference time-domain (FDTD) method, the simulation results demonstrate that both large S of 200 nm/RIU (RIU=refractive index unit) and high Q-factor >104 at telecom wavelength range can be achieved simultaneously. And the sensor figure of merit (FOM)>7000 is featured, an order of magnitude improvement over previous 2D PC sensors array. In addition, for the proposed 2D PC-ISA device, each sensor unit is shown to independently shift its resonance wavelength in response to the changes in refractive index (RI) and does not perturb the others. Thus, it is potentially an ideal platform for realizing ultra-compact lab-on-a-chip applications with dense arrays of functionalized spots for multiplexed sensing, and also can be used as an opto-fluidic architecture for performing highly parallel detection of biochemical interactions in aqueous environments.

  12. Viral RNA modulates the acid sensitivity of foot-and-mouth disease virus capsids.

    PubMed Central

    Curry, S; Abrams, C C; Fry, E; Crowther, J C; Belsham, G J; Stuart, D I; King, A M

    1995-01-01

    Foot-and-mouth disease virus (FMDV) manifests an extreme sensitivity to acid, which is thought to be important for entry of the RNA genome into the cell. We have compared the low-pH-induced disassembly in vitro of virions and natural empty capsids of three subtypes of serotype A FMDV by enzyme-linked immunosorbent assay and sucrose gradient sedimentation analysis. For all three subtypes (A22 Iraq 24/64, A10(61), and A24 Cruzeiro), the empty capsid was more stable by 0.5 pH unit on average than the corresponding virion. Unexpectedly, in the natural empty capsids used in this study, the precursor capsid protein VP0 was found largely to be cleaved into VP2 and VP4. For picornaviruses the processing of VP0 is closely associated with encapsidation of viral RNA, which is considered likely to play a catalytic role in the cleavage. Investigation of the cleavage of VP0 in natural empty capsids failed to implicate the viral RNA. However, it remains possible that these particles arise from abortive attempts to encapsidate RNA. Empty capsids expressed from a vaccinia virus recombinant showed essentially the same acid lability as natural empty capsids, despite differing considerably in the extent of VP0 processing, with the synthetic particles containing almost exclusively uncleaved VP0. These results indicate that it is the viral RNA that modulates acid lability in FMDV. In all cases the capsids dissociate at low pH directly into pentameric subunits. Comparison of the three viruses indicates that FMDV A22 Iraq is about 0.5 pH unit more sensitive to low pH than types A10(61) and A24 Cruzeiro. Sequence analysis of the three subtypes identified several differences at the interface between pentamers and highlighted a His-alpha-helix dipole interaction which spans the pentamer interface and appears likely to influence the acid lability of the virus. PMID:7983739

  13. Sensitive quantitative detection of commensal bacteria by rRNA-targeted reverse transcription-PCR.

    PubMed

    Matsuda, Kazunori; Tsuji, Hirokazu; Asahara, Takashi; Kado, Yukiko; Nomoto, Koji

    2007-01-01

    A sensitive rRNA-targeted reverse transcription-quantitative PCR (RT-qPCR) method was developed for exact and sensitive enumeration of subdominant bacterial populations. Using group- or species-specific primers for 16S or 23S rRNA, analytical curves were constructed for Escherichia coli, Enterococcus faecalis, Staphylococcus aureus, Clostridium perfringens, and Pseudomonas aeruginosa, and the threshold cycle value was found to be linear up to an RNA amount of 10(-3) cell per RT-PCR. The number of bacteria in culture was determined by RT-qPCR, and the results correlated well with the CFU count over the range from 10(0) to 10(5) CFU. The bacterial counts obtained by RT-qPCR were the same as the CFU counts irrespective of the growth phase in vitro, except for C. perfringens during starvation periods; the viable cell counts obtained by using a combination of 4',6-diamidino-2-phenylindole (DAPI) staining and SYTO9-propidium iodide double staining were in good agreement with the RT-qPCR counts rather than with the CFU counts. The RT-qPCR method could detect endogenous Enterobacteriaceae and P. aeruginosa in feces of hospitalized patients (n = 38) at a level of 10(3) cells per g of feces, and for enumeration of S. aureus or P. aeruginosa spiked into human peripheral blood, the lower detection limit for RT-qPCR quantification of the bacteria was 2 cells per ml of blood, suggesting that this method was equivalent to the conventional culture method. As only 5 h was needed for RT-qPCR quantification, we suggest that rRNA-targeted RT-qPCR assays provide a sensitive and convenient system for quantification of commensal bacteria and for examining their possible invasion of a host.

  14. Enantiodifferentiating Z-E photoisomerization of cyclooctene sensitized by DNA and RNA.

    PubMed

    Sugahara, N; Kawano, M; Wada, T; Inoue, Y

    2000-01-01

    DNA and RNA have been shown for the first time to function as chiral photosensitizers in aqueous solution, to effect the enantiodifferentiating photoisomerization of (Z)-cyclooctene (1Z), giving the chiral (E)-isomer in enantiomeric excesses (ee's) of up to 15%. In order to elucidate the effect of nucleotide sequence, enantiodifferentiating photoisomerization of 1Z was also performed using oligo and homopolynucleotides as chiral sensitizer. The -18.8% ee was observed by using d(T)15.d(A)15 as sensitizer, whereas sensitization by the poly(U).poly(A) duplex gave only racemic (E)-cyclooctene. From these results, oligothymidine sequence is essential for efficient enantioselective photoisomerization of 1Z.

  15. A highly sensitive and selective viral protein detection method based on RNA oligonucleotide nanoparticle

    PubMed Central

    Roh, Changhyun; Lee, Ho-Young; Kim, Sang-Eun; Jo, Sung-Kee

    2010-01-01

    Globally, approximately 170 million people (representing approximately 3% of the population worldwide), are infected with hepatitis C virus (HCV) and at risk of serious liver disease, including chronic hepatitis. We propose a new quantum dots (QDs)-supported RNA oligonucleotide approach for the specific and sensitive detection of viral protein using a biochip. This method was developed by immobilizing a HCV nonstructural protein 5B (NS5B) on the surface of a glass chip via the formation of a covalent bond between an amine protein group and a ProLinker™ glass chip. The QDs-supported RNA oligonucleotide was conjugated via an amide formation reaction from coupling of a 5′-end-amine-modified RNA oligonucleotide on the surface of QDs displaying carboxyl groups via standard EDC coupling. The QDs-conjugated RNA oligonucleotide was interacted to immobilized viral protein NS5B on the biochip. The detection is based on the variation of signal of QDs-supported RNA oligonucleotide bound on an immobilized biochip. It was demonstrated that the value of the signal has a linear relationship with concentrations of the HCV NS5B viral protein in the 1 μg mL−1 to 1 ng mL−1 range with a detection limit of 1 ng mL−1. The major advantages of this RNA-oligonucleotide nanoparticle assay are its good specificity, ease of performance, and ability to perform one-spot monitoring. The proposed method could be used as a general method of HCV detection and is expected to be applicable to other types of diseases as well. PMID:20517476

  16. Sensitivity to Alternaria alternata toxin in citrus because of altered mitochondrial RNA processing

    PubMed Central

    Ohtani, Kouhei; Yamamoto, Hiroyuki; Akimitsu, Kazuya

    2002-01-01

    Specificity in the interaction between rough lemon (Citrus jambhiri Lush.) and the fungal pathogen Alternaria alternata rough lemon pathotype is determined by a host-selective toxin, ACR-toxin. Mitochondria from rough lemon are sensitive to ACR-toxin whereas mitochondria from resistant plants, including other citrus species, are resistant. We have identified a C. jambhiri mitochondrial DNA sequence, designated ACRS (ACR-toxin sensitivity gene), that confers toxin sensitivity to Escherichia coli. ACRS is located in the group II intron of the mitochondrial tRNA-Ala and is translated into a SDS-resistant oligomeric protein in C. jambhiri mitochondria but is not translated in the toxin-insensitive mitochondria. ACRS is present in the mitochondrial genome of both toxin-sensitive and -insensitive citrus. However, in mitochondria of toxin-insensitive plants, the transcripts from ACRS are shorter than those in mitochondria of sensitive plants. These results demonstrate that sensitivity to ACR-toxin and hence specificity of the interaction between A. alternata rough lemon pathotype and C. jambhiri is due to differential posttranscriptional processing of a mitochondrial gene. PMID:11842194

  17. Sensitive Detection Assays for Influenza RNA Do Not Reveal Viremia in US Blood Donors

    PubMed Central

    Stramer, Susan L.; Collins, Cynthia; Nugent, Thomas; Wang, Xue; Fuschino, Meghan; Heitman, John W.; Law, Jacqueline; Krysztof, David E.; Kiely, Nancy; Todd, Deborah; Vermeulen, Nicolaas M. J.; Harrington, Karen; Kamel, Hany; Kelvin, David J.; Busch, Michael P.; St. George, Kirsten; Hewlett, Indira K.; Linnen, Jeffrey M.

    2012-01-01

    (See the article by Delwart et al, on pages 875–85, and see the editorial commentary by Katz, on pages 867–9.) Background. There have been anecdotal reports of influenza viremia since the 1960s. We present an assessment of the prevalence of seasonal and 2009 H1N1 influenza viremia (via RNA testing) in blood donor populations using multiple sensitive detection assays. Methods. Several influenza RNA amplification assays, including transcription-mediated amplification (TMA) and 2 reverse-transcription polymerase chain reaction (RT-PCR) assays, were evaluated and used to test donor samples. Retrospective samples from 478 subjects drawn at sites with high influenza activity were tested. Prospective samples were collected from 1004 blood donors who called their donation center within 3 days of donation complaining of influenza-like illness (ILI). The plasma collected on the day of donation for these subjects was tested. Results. Of the repository samples, 2 of 478 plasma samples were initially reactive but not repeat reactive by influenza TMA. Of blood donors reporting ILI symptoms postdonation, 1 of 1004 samples was TMA initially reactive but not repeat reactive; all samples were nonreactive by RT-PCR testing. Conclusions. Targeting blood donor populations most likely to have influenza infection, we failed to detect influenza RNA in 1482 donor samples, with most tested by 3 different RNA assays. Seasonal influenza does not appear to pose a significant contamination threat to the blood supply. PMID:22293429

  18. Direct observation of mobility state transitions in RNA trajectories by sensitive single molecule feedback tracking

    PubMed Central

    Spille, Jan-Hendrik; Kaminski, Tim P.; Scherer, Katharina; Rinne, Jennifer S.; Heckel, Alexander; Kubitscheck, Ulrich

    2015-01-01

    Observation and tracking of fluorescently labeled molecules and particles in living cells reveals detailed information about intracellular processes on the molecular level. Whereas light microscopic particle observation is usually limited to two-dimensional projections of short trajectory segments, we report here image-based real-time three-dimensional single particle tracking in an active feedback loop with single molecule sensitivity. We tracked particles carrying only 1–3 fluorophores deep inside living tissue with high spatio-temporal resolution. Using this approach, we succeeded to acquire trajectories containing several hundred localizations. We present statistical methods to find significant deviations from random Brownian motion in such trajectories. The analysis allowed us to directly observe transitions in the mobility of ribosomal (r)RNA and Balbiani ring (BR) messenger (m)RNA particles in living Chironomus tentans salivary gland cell nuclei. We found that BR mRNA particles displayed phases of reduced mobility, while rRNA particles showed distinct binding events in and near nucleoli. PMID:25414330

  19. RNase non-sensitive and endocytosis independent siRNA delivery system: delivery of siRNA into tumor cells and high efficiency induction of apoptosis

    NASA Astrophysics Data System (ADS)

    Jiang, Xinglu; Wang, Guobao; Liu, Ru; Wang, Yaling; Wang, Yongkui; Qiu, Xiaozhong; Gao, Xueyun

    2013-07-01

    To date, RNase degradation and endosome/lysosome trapping are still serious problems for siRNA-based molecular therapy, although different kinds of delivery formulations have been tried. In this report, a cell penetrating peptide (CPP, including a positively charged segment, a linear segment, and a hydrophobic segment) and a single wall carbon nanotube (SWCNT) are applied together by a simple method to act as a siRNA delivery system. The siRNAs first form a complex with the positively charged segment of CPP via electrostatic forces, and the siRNA-CPP further coats the surface of the SWCNT via hydrophobic interactions. This siRNA delivery system is non-sensitive to RNase and can avoid endosome/lysosome trapping in vitro. When this siRNA delivery system is studied in Hela cells, siRNA uptake was observed in 98% Hela cells, and over 70% mRNA of mammalian target of rapamycin (mTOR) is knocked down, triggering cell apoptosis on a significant scale. Our siRNA delivery system is easy to handle and benign to cultured cells, providing a very efficient approach for the delivery of siRNA into the cell cytosol and cleaving the target mRNA therein.

  20. SYBR Gold Fluorescence Quenching is a Sensitive Probe of Chitosan-microRNA Interactions.

    PubMed

    Santos-Carballal, Beatriz; Swamy, Musti J; Moerschbacher, Bruno M; Goycoolea, Francisco M

    2016-01-01

    Competitive dye displacement titration has previously been used to characterize chitosan-DNA interactions using ethidium bromide. In this work, we aim to develop a fast and reliable method using SYBR Gold as a fluorescent probe to evaluate the binding affinity between ssRNA and chitosan. The interaction of chitosan with ssRNA was investigated as a function of temperature, molecular weight and degree of acetylation of chitosan, using competitive dye displacement titrations with fluorescence quenching. Affinity constants are reported, showing the high sensitivity of the interaction to the degree of acetylation of chitosan and barely dependent on the molecular weight. We propose that the mechanism of SYBR Gold fluorescence quenching is governed by both static and dynamic quenching.

  1. Clinical Impact of a Novel MicroRNA Chemo-Sensitivity Predictor in Gastrooesophageal Cancer

    PubMed Central

    Winther, Mette; Knudsen, Steen; Dahlgaard, Jesper; Jensen, Thomas; Hansen, Anker; Jensen, Peter Buhl; Tramm, Trine; Alsner, Jan; Nordsmark, Marianne

    2016-01-01

    Background miRNAs might be potentially useful biomarkers for prediction of response to chemotherapeutic agents, radiotherapy and survival. The aim of this retrospective study was to validate miRNA response predictors in a cohort of patients with gastrooesophageal cancer in order to predict overall survival (OS) and disease-specific survival (DSS). Material and Methods The study population encompassed 53 patients treated with curative intend for loco-regional gastrooesophageal cancer. miRNA expression was quantified from pre-therapeutic and diagnostic, formalin-fixed, paraffin embedded tumour specimens using Affymetrix GeneChip miRNA 1.0 Array. Based on growth inhibition of the NCI60 panel in the presence of cisplatin, epirubicine and capecitabine, a miRNA based response predictor was developed. The Cox proportional hazards model was applied to assess the correlations of the response predictor with OS and DSS. Results A univariate analysis demonstrated a statistical significant improvement of OS for patients who had undergone surgical resection with prediction scores above the median prediction score (HR: 0.41 (95% CI: 0.17–0.96). Adjusting for surgery and stage, this predictor was identified to be independently associated with both OS (HR: 0.37 (95% CI: 0.16–0.87)) and DSS (HR: 0.32 (0.12–0.87)). Conclusion The miRNA profile predictive for sensitivity to cisplatin, epirubicine and capecitabine was shown to be independently associated with OS and DSS in patients with gastrooesophageal cancer. PMID:26885979

  2. Antibody array in a multiwell plate format for the sensitive and multiplexed detection of important plant pathogens.

    PubMed

    Charlermroj, Ratthaphol; Himananto, Orawan; Seepiban, Channarong; Kumpoosiri, Mallika; Warin, Nuchnard; Gajanandana, Oraprapai; Elliott, Christopher T; Karoonuthaisiri, Nitsara

    2014-07-15

    The global seed market is considered to be an important industry with a total value of $10,543 million US dollars in 2012. Because plant pathogens such as bacteria and viruses cause a significant economic loss to both producers and exporters, the seed export industry urgently requires rapid, sensitive, and inexpensive testing for the pathogens to prevent disease spreading worldwide. This study developed an antibody array in a multiwell plate format to simultaneously detect four crucial plant pathogens, namely, a bacterial fruit blotch bacterium Acidovorax avenae subsp. citrulli (Aac), Chilli veinal mottle virus (ChiVMV, potyvirus), Watermelon silver mottle virus (WSMoV, tospovirus serogroup IV), and Melon yellow spot virus (MYSV, tospovirus). The capture antibodies specific to the pathogens were immobilized on each well at preassigned positions by an automatic microarrayer. The antibodies on the arrays specifically captured the corresponding pathogens present in the sample extracts. The presence of pathogens bound on the capture antibodies was subsequently detected by a cocktail of fluorescently conjugated secondary antibodies. The limits of detection of the developed antibody array for the detection of Aac, ChiVMV, WSMoV, and MYSV were 5 × 10(5) CFU/mL, 30 ng/mL, 1000 ng/mL, and 160 ng/mL, respectively, which were very similar to those of the conventional ELISA method. The antibody array in a multiwell plate format accurately detected plant pathogens in single and multiple detections. Moreover, this format enables easy handling of the assay at a higher speed of operation. PMID:24945525

  3. Rapid discrimination of rabies viruses isolated from various host species in Brazil by multiplex reverse transcription-polymerase chain reaction.

    PubMed

    Sato, Go; Tanabe, Hitomi; Shoji, Youko; Itou, Takuya; Ito, Fumio H; Sato, Tetsuo; Sakai, Takeo

    2005-08-01

    Rabies is carried mainly by mammalian carnivores and vampire bats in Latin America. However, rabies virus (RV) has been isolated in recent years from not only vampire bats in rural areas but also from several non-vampire bat species in urban areas, respectively. Therefore, rapid molecular screening is necessary for efficient epidemiology of these RVs. In this study, we investigated the usefulness of multiplex reverse transcription-polymerase chain reaction (RT-PCR) for determining the origins of 54 RV isolates from various host species in Brazil. And to evaluate the multiplex RT-PCR as a potential diagnostic tool, we investigated the sensitivity of this method. In addition, we compared the results with a phylogenetic tree developed from sequences of the RV glycoprotein (G protein) gene. Multiplex RT-PCR products showed five different sizes of products, whereas the phylogenic tree showed six groups. Of these six groups, four corresponded with the four sizes of the multiplex RT-PCR products. The other two groups showed correspondance with another one size of the multiplex RT-PCR products, indicating that multiplex RT-PCR results reflected the lineage of the 54 isolates. This study also showed that this method can detect trace amounts of RNA. In conclusion, this multiplex RT-PCR method allows the rapid, specific, and simultaneous detection of RVs isolated from various host species in Brazil. PMID:16036175

  4. Isothermal sensitive detection of microRNA using an autonomous DNA machine recycling output as input.

    PubMed

    Ogawa, Atsushi

    2010-10-15

    An autonomous DNA machine recycling the output as the input for isothermal, sensitive, and specific detection of miRNAs has been developed. This machine shows considerably high signal amplification efficiency (∼1000-fold) and thus a low detection limit (∼20 amol). The machine also shows high specificity, discriminating 50 amol of synthetic miRNA from 100-fold larger amounts of its family member and from 100 ng of unrelated total RNAs. Moreover, it is available for practically detecting natural miRNAs in total RNAs.

  5. Sensitive Multiplexed Quantitative Analysis of Autoantibodies to Cancer Antigens with Chemically S-Cationized Full-Length and Water-Soluble Denatured Proteins.

    PubMed

    Futami, Junichiro; Nonomura, Hidenori; Kido, Momoko; Niidoi, Naomi; Fujieda, Nao; Hosoi, Akihiro; Fujita, Kana; Mandai, Komako; Atago, Yuki; Kinoshita, Rie; Honjo, Tomoko; Matsushita, Hirokazu; Uenaka, Akiko; Nakayama, Eiichi; Kakimi, Kazuhiro

    2015-10-21

    Humoral immune responses against tumor-associated antigens (TAAs) or cancer/testis antigens (CTAs) aberrantly expressed in tumor cells are frequently observed in cancer patients. Recent clinical studies have elucidated that anticancer immune responses with increased levels of anti-TAA/CTA antibodies improve cancer survival rates. Thus, these antibody levels are promising biomarkers for diagnosing the efficiency of cancer immunotherapy. Full-length antigens are favored for detecting anti-TAA/CTA antibodies because candidate antigen proteins contain multiple epitopes throughout their structures. In this study, we developed a methodology to prepare purified water-soluble and full-length antigens by using cysteine sulfhydryl group cationization (S-cationization) chemistry. S-Cationized antigens can be prepared from bacterial inclusion bodies, and they exhibit improved protein solubility but preserved antigenicity. Anti-TAA/CTA antibodies detected in cancer patients appeared to recognize linear epitopes, as well as conformational epitopes, and because the frequency of cysteine side-residues on the epitope-paratope interface was low, any adverse effects of S-cationization were virtually negligible for antibody binding. Furthermore, S-cationized antigen-immobilized Luminex beads could be successfully used in highly sensitive quantitative-multiplexed assays. Indeed, patients with a more broadly induced serum anti-TAA/CTA antibody level showed improved progression-free survival after immunotherapy. The comprehensive anti-TAA/CTA assay system, which uses S-cationized full-length and water-soluble recombinant antigens, may be a useful diagnostic tool for assessing the efficiency of cancer immunotherapy. PMID:26355635

  6. Multiplex, Rapid, and Sensitive Isothermal Detection of Nucleic-Acid Sequence by Endonuclease Restriction-Mediated Real-Time Multiple Cross Displacement Amplification

    PubMed Central

    Wang, Yi; Wang, Yan; Zhang, Lu; Liu, Dongxin; Luo, Lijuan; Li, Hua; Cao, Xiaolong; Liu, Kai; Xu, Jianguo; Ye, Changyun

    2016-01-01

    We have devised a novel isothermal amplification technology, termed endonuclease restriction-mediated real-time multiple cross displacement amplification (ET-MCDA), which facilitated multiplex, rapid, specific and sensitive detection of nucleic-acid sequences at a constant temperature. The ET-MCDA integrated multiple cross displacement amplification strategy, restriction endonuclease cleavage and real-time fluorescence detection technique. In the ET-MCDA system, the functional cross primer E-CP1 or E-CP2 was constructed by adding a short sequence at the 5′ end of CP1 or CP2, respectively, and the new E-CP1 or E-CP2 primer was labeled at the 5′ end with a fluorophore and in the middle with a dark quencher. The restriction endonuclease Nb.BsrDI specifically recognized the short sequence and digested the newly synthesized double-stranded terminal sequences (5′ end short sequences and their complementary sequences), which released the quenching, resulting on a gain of fluorescence signal. Thus, the ET-MCDA allowed real-time detection of single or multiple targets in only a single reaction, and the positive results were observed in as short as 12 min, detecting down to 3.125 fg of genomic DNA per tube. Moreover, the analytical specificity and the practical application of the ET-MCDA were also successfully evaluated in this study. Here, we provided the details on the novel ET-MCDA technique and expounded the basic ET-MCDA amplification mechanism. PMID:27242766

  7. Knockdown of long noncoding RNA H19 sensitizes human glioma cells to temozolomide therapy.

    PubMed

    Jiang, Pengfei; Wang, Ping; Sun, Xiaoling; Yuan, Zhongshun; Zhan, Rucai; Ma, Xiangyu; Li, Weiguo

    2016-01-01

    Temozolomide (TMZ) is commonly used in glioma chemotherapy. However, a great clinical challenge for TMZ is chemoresistance. H19 transcripts are recognized as long noncoding RNAs, which potentially interact with chromatin-modifying complexes to regulate gene expression via epigenetic changes. Our data based on glioma patients showed that the expression of H19 was significantly upregulated in TMZ-resistant tumors compared with the TMZ-sensitive tumors. To determine the function of H19 in glioma, cell lines U87 and U251 were exposed to TMZ to establish TMZ-resistant clones U87(TMZ) and U251(TMZ). In U87(TMZ) and U251(TMZ), the expression level of H19 transcripts was increased compared to wild-type or nonresistant clones, as determined by real-time quantitative reverse transcription polymerase chain reaction. Concomitant treatment with small interfering RNA specifically targeting H19 and TMZ in resistant glioma clones resulted in decreased IC50 values for TMZ, and increased apoptotic rates than control small interfering RNA-treated cells. This was also evident by the increased PARP cleavage in resistant cells exposed to TMZ + si-H19. Furthermore, the reduced expression of H19 altered major drug resistance genes, such as MDR, MRP, and ABCG2, both at the mRNA and protein levels. Taken together, these findings suggest that H19 plays an important role in the development of TMZ resistance, and may represent a novel therapeutic target for TMZ-resistant gliomas. PMID:27366087

  8. Knockdown of long noncoding RNA H19 sensitizes human glioma cells to temozolomide therapy

    PubMed Central

    Jiang, Pengfei; Wang, Ping; Sun, Xiaoling; Yuan, Zhongshun; Zhan, Rucai; Ma, Xiangyu; Li, Weiguo

    2016-01-01

    Temozolomide (TMZ) is commonly used in glioma chemotherapy. However, a great clinical challenge for TMZ is chemoresistance. H19 transcripts are recognized as long noncoding RNAs, which potentially interact with chromatin-modifying complexes to regulate gene expression via epigenetic changes. Our data based on glioma patients showed that the expression of H19 was significantly upregulated in TMZ-resistant tumors compared with the TMZ-sensitive tumors. To determine the function of H19 in glioma, cell lines U87 and U251 were exposed to TMZ to establish TMZ-resistant clones U87TMZ and U251TMZ. In U87TMZ and U251TMZ, the expression level of H19 transcripts was increased compared to wild-type or nonresistant clones, as determined by real-time quantitative reverse transcription polymerase chain reaction. Concomitant treatment with small interfering RNA specifically targeting H19 and TMZ in resistant glioma clones resulted in decreased IC50 values for TMZ, and increased apoptotic rates than control small interfering RNA-treated cells. This was also evident by the increased PARP cleavage in resistant cells exposed to TMZ + si-H19. Furthermore, the reduced expression of H19 altered major drug resistance genes, such as MDR, MRP, and ABCG2, both at the mRNA and protein levels. Taken together, these findings suggest that H19 plays an important role in the development of TMZ resistance, and may represent a novel therapeutic target for TMZ-resistant gliomas. PMID:27366087

  9. MicroRNA-9 suppresses the sensitivity of CNE2 cells to ultraviolet radiation.

    PubMed

    Zheng, Chao-Pan; Han, Ling; Hou, Wei-Jian; Tang, Jun; Wen, Yi-Hui; Fu, Ran; Wang, Yue-Jian; Wen, Wei-Ping

    2015-08-01

    MicroRNA (miR)-9 has been demonstrated to regulate the radiosensitivity of tumor cells. In the present study, the mechanism by which miR-9 modulates the sensitivity of nasopharyngeal carcinoma (NPC) cells to ultraviolet (UV) radiation was investigated. The results demonstrated that exposure of NPC cells to UV light resulted in a significant increase in the expression of miR-9, and that CNE2 cells overexpressing miR-9 exhibited reduced levels of DNA damage and increased levels of total glutathione upon UV exposure. Accordingly, the inhibition of the expression of miR-9 promoted UV-induced DNA damage and apoptosis. Although miR-9 inhibited the expression of E-cadherin in the CNE2 cells and increased their resistance to UV radiation, the use of small interfering RNA to inhibit the expression of E-cadherin was not sufficient to decrease the radiosensitivity of the NPC cells. These data demonstrated that miR-9 did not modulate the sensitivity of the CNE2 cells to UV radiation through E-cadherin, but suggested that miR-9 regulated radiosensitivity through its effects on glutathione. These findings suggest that miR-9 may be a potential target for modulating the radiosensitivity of NPC cells.

  10. Second harmonic super-resolution microscopy for quantification of mRNA at single copy sensitivity.

    PubMed

    Liu, Jing; Cho, Il-Hoon; Cui, Yi; Irudayaraj, Joseph

    2014-12-23

    Cell-specific information on the quantity and localization of key mRNAs at single copy sensitivity in single cells is critical for evaluating basic cellular process, disease risk, and efficacy of therapy. Quantification of overexpressed mRNAs beyond the diffraction limit is constrained by the optical property of the probes and microscopy techniques. In this report, nanosized barium titanium oxide (BaTiO3, BTO) crystals were utilized as probes for mRNA quantification by a second harmonic super-resolution microscopy (SHaSM). The SHaSM was able to detect a single copy of the human epidermal growth factor receptor 2 (Her2) mRNA at a resolution of 55.6 nm with the ability to resolve multiple mRNA copies in a diffraction-limited spot. Her2 mRNA per cell was counted in SK-BR-3, MCF-7, and HeLa cell lines as 595±79.1, 38.9±8.26, and 1.5±2.8, respectively. Our single-cell quantification results were validated with the fluorescence in situ hybridization studies and quantitative PCR, showing better specificity and selectivity over current single-molecule approaches for transcript detection. The SHaSM is expected to have an upper limit of resolving ∼10(4) transcripts in a single cell with the ability to monitor intracellular transcriptional dynamics at video rate. The developed approach has strong potential in clinical research and in the early diagnosis of life-threatening diseases such as cancer.

  11. A long noncoding RNA regulates photoperiod-sensitive male sterility, an essential component of hybrid rice.

    PubMed

    Ding, Jihua; Lu, Qing; Ouyang, Yidan; Mao, Hailiang; Zhang, Pingbo; Yao, Jialing; Xu, Caiguo; Li, Xianghua; Xiao, Jinghua; Zhang, Qifa

    2012-02-14

    Hybrid rice has greatly contributed to the global increase of rice productivity. A major component that facilitated the development of hybrids was a mutant showing photoperiod-sensitive male sterility (PSMS) with its fertility regulated by day length. Transcriptome studies have shown that large portions of the eukaryotic genomic sequences are transcribed to long noncoding RNAs (lncRNAs). However, the potential roles for only a few lncRNAs have been brought to light at present. Thus, great efforts have to be invested to understand the biological functions of lncRNAs. Here we show that a lncRNA of 1,236 bases in length, referred to as long-day-specific male-fertility-associated RNA (LDMAR), regulates PSMS in rice. We found that sufficient amount of the LDMAR transcript is required for normal pollen development of plants grown under long-day conditions. A spontaneous mutation causing a single nucleotide polymorphism (SNP) between the wild-type and mutant altered the secondary structure of LDMAR. This change brought about increased methylation in the putative promoter region of LDMAR, which reduced the transcription of LDMAR specifically under long-day conditions, resulting in premature programmed cell death (PCD) in developing anthers, thus causing PSMS. Thus, a lncRNA could directly exert a major effect on a trait like a structure gene, and a SNP could alter the function of a lncRNA similar to amino acid substitution in structural genes. Molecular elucidating of PSMS has important implications for understanding molecular mechanisms of photoperiod regulation of many biological processes and also for developing male sterile germplasms for hybrid crop breeding.

  12. A long noncoding RNA regulates photoperiod-sensitive male sterility, an essential component of hybrid rice

    PubMed Central

    Ding, Jihua; Lu, Qing; Ouyang, Yidan; Mao, Hailiang; Zhang, Pingbo; Yao, Jialing; Xu, Caiguo; Li, Xianghua; Xiao, Jinghua; Zhang, Qifa

    2012-01-01

    Hybrid rice has greatly contributed to the global increase of rice productivity. A major component that facilitated the development of hybrids was a mutant showing photoperiod-sensitive male sterility (PSMS) with its fertility regulated by day length. Transcriptome studies have shown that large portions of the eukaryotic genomic sequences are transcribed to long noncoding RNAs (lncRNAs). However, the potential roles for only a few lncRNAs have been brought to light at present. Thus, great efforts have to be invested to understand the biological functions of lncRNAs. Here we show that a lncRNA of 1,236 bases in length, referred to as long-day–specific male-fertility–associated RNA (LDMAR), regulates PSMS in rice. We found that sufficient amount of the LDMAR transcript is required for normal pollen development of plants grown under long-day conditions. A spontaneous mutation causing a single nucleotide polymorphism (SNP) between the wild-type and mutant altered the secondary structure of LDMAR. This change brought about increased methylation in the putative promoter region of LDMAR, which reduced the transcription of LDMAR specifically under long-day conditions, resulting in premature programmed cell death (PCD) in developing anthers, thus causing PSMS. Thus, a lncRNA could directly exert a major effect on a trait like a structure gene, and a SNP could alter the function of a lncRNA similar to amino acid substitution in structural genes. Molecular elucidating of PSMS has important implications for understanding molecular mechanisms of photoperiod regulation of many biological processes and also for developing male sterile germplasms for hybrid crop breeding. PMID:22308482

  13. Increased sensitivity of RT-PCR for Potato virus Y detection using RNA isolated by a procedure with differential centrifugation.

    PubMed

    Zhang, Jianhua; Nie, Xianzhou; Boquel, Sébastien; Al-Daoud, Fadi; Pelletier, Yvan

    2015-12-01

    The sensitivity of reverse transcription-polymerase chain reaction (RT-PCR) for virus detection is influenced by many factors such as specificity of primers and quality of templates. These factors become extremely important for successful detection when virus concentration is low. Total RNA isolated from Potato virus Y (PVY)-infected potato plants using the sodium sulfite RNA isolation method or RNeasy plant mini kit contains a high proportion of host RNA and may also contain trace amount of phenolic and polysaccharide residues, which may inhibit RT-PCR. The goal of this study was to enhance the sensitivity of PVY detection by reducing host RNA in the extract by differential centrifugation followed by extraction using an RNeasy mini kit (DCR method). One-step RT-PCR had relatively low amplification efficiency for PVY RNA when a high proportion of plant RNA was present. SYBR Green-based real time RT-PCR showed that the RNA isolated by the DCR method had a higher cycle threshold value (Ct) for the elongation factor 1-α mRNA (Ef1α) of potato than the Ct value of the RNA extracted using the RNeasy plant mini kit, indicating that the DCR method significantly reduced the proportion of potato RNA in the extract. The detectable amount of RNA extracted using the DCR method was <0.001ng when plant sap from 10 PVY-infected and PVY-free potato leaflets in a 1.5:100 fresh weight ratio was extracted, compared with 0.01 and 0.02ng of RNA using the RNeasy plant mini kit and sodium sulfite RNA isolation methods, respectively. PMID:26210699

  14. 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…

  15. Cold-Sensitive Pseudomonas RNA Polymerase I. Characterization of the Host Dependent Cold-Sensitive Restriction of Phage CB3

    PubMed Central

    Sobieski, Rodney J.; Olsen, Ronald H.

    1973-01-01

    Analysis of bacteriophage CB3 infection of Pseudomonas aeruginosa strain PAT2 establishes that phage induced changes in net macromolecular synthesis are absent at nonpermissive phage growth temperatures (32 C). Alterations which are evident in the PAT2 strain at 37 C or in the fully permissive strain, PAO1C, at either warm or cold temperatures do not occur in PAT2 at low temperatures. CB3 DNA synthesis and the degradation of host DNA to approximately 78S components occur at 37 C, but are absent in PAT2 at 20 C. Nevertheless, attachment of phage DNA to host cytoplasmic material occurs under permissive and nonpermissive conditions. This binding of phage DNA at 20 C is identical in nature to phage DNA bound at 37 C. Thus, the conditional cold-sensitive PAT2 host function in the growth of CB3 is expressed subsequent to membrane binding of the infecting genomes but prior to the onset of the initiation of CB3 DNA synthesis, the inhibition of host DNA synthesis, and the transient depression in RNA synthesis which occurs in permissive cells. PMID:4202617

  16. Multiplex quantitative foodborne pathogen detection using high resolution CE-SSCP coupled stuffer-free multiplex ligation-dependent probe amplification.

    PubMed

    Chung, Boram; Shin, Gi Won; Na, Jeongkyeong; Oh, Mi-Hwa; Jung, Gyoo Yeol

    2012-05-01

    Sensitive multiplex detection methods for foodborne pathogens are important in controlling food safety, and detection of genetic markers is accepted to be one of the best tools for sensitive detection. Although CE technology offers great potential in terms of sensitive multiplex detection, the necessary amplification is confined to markers sharing common primers such as the 16S rRNA gene. For precise and sensitive detection, pathogen-specific genes are optimal markers. Although multiplex ligation-dependent probe amplification (MLPA) is appropriate for amplification of specific markers, the requirement for stuffers, to ensure length-dependent separation on CE, is a major obstacle in detection of foodborne pathogens. In the present study, we developed stuffer-free MLPA using high-resolution CE-SSCP to sensitively detect ten foodborne pathogens. The probe set for MLPA prior to CE-SSCP analysis was designed for species-specific detection. After careful optimization of each MLPA step, to ensure that CE-SSCP analysis was informative, we found that all ten pathogens could be reliably identified; the limits of detection were 0.5-5 pg of genomic DNA, and more than 100-fold increase could be quantitatively determined. Thus, MLPA-CE-SSCP is a sensitive and reliable technique for pathogen detection.

  17. Multiplex Real-Time PCR Assay for Rapid Detection of Methicillin-Resistant Staphylococci Directly from Positive Blood Cultures

    PubMed Central

    Wang, Hye-young; Kim, Sunghyun; Kim, Jungho; Park, Soon-Deok

    2014-01-01

    Methicillin-resistant Staphylococcus aureus (MRSA) is the most prevalent cause of bloodstream infections (BSIs) and is recognized as a major nosocomial pathogen. This study aimed to evaluate a newly designed multiplex real-time PCR assay capable of the simultaneous detection of mecA, S. aureus, and coagulase-negative staphylococci (CoNS) in blood culture specimens. The Real-MRSA and Real-MRCoNS multiplex real-time PCR assays (M&D, Republic of Korea) use the TaqMan probes 16S rRNA for Staphylococcus spp., the nuc gene for S. aureus, and the mecA gene for methicillin resistance. The detection limit of the multiplex real-time PCR assay was 103 CFU/ml per PCR for each gene target. The multiplex real-time PCR assay was evaluated using 118 clinical isolates from various specimen types and a total of 350 positive blood cultures from a continuous monitoring blood culture system. The results obtained with the multiplex real-time PCR assay for the three targets were in agreement with those of conventional identification and susceptibility testing methods except for one organism. Of 350 positive bottle cultures, the sensitivities of the multiplex real-time PCR kit were 100% (166/166 cultures), 97.2% (35/36 cultures), and 99.2% (117/118 cultures) for the 16S rRNA, nuc, and mecA genes, respectively, and the specificities for all three targets were 100%. The Real-MRSA and Real-MRCoNS multiplex real-time PCR assays are very useful for the rapid accurate diagnosis of staphylococcal BSIs. In addition, the Real-MRSA and Real-MRCoNS multiplex real-time PCR assays could have an important impact on the choice of appropriate antimicrobial therapy, based on detection of the mecA gene. PMID:24648566

  18. RNA-seq identifies determinants of oxaliplatin sensitivity in colorectal cancer cell lines.

    PubMed

    Li, Xin-Xiang; Peng, Jun-Jie; Liang, Lei; Huang, Li-Yong; Li, Da-Wei; Shi, De-Bing; Zheng, Hong-Tu; Cai, San-Jun

    2014-01-01

    Oxaliplatin-based chemotherapy, such as FOLFOX, is the first-line therapy for advanced colorectal cancer (CRC) or metastatic CRC patients. However, the partial response of patients to these regimes and the severe peripheral neuropathy toxicity induced by oxaliplatin makes it urgent to figure out biomarkers for oxaliplatin sensitivity to select suitable patients who benefit from these treatments. In present work, 21 CRC cell lines with different sensitivities to oxaliplatin were applied to RNA-seq. The basal expression profiles of these cell lines were correlated to their response to oxaliplatin. Bioinformatics analysis suggested that expression of 58 genes was correlated, negatively or positively, to oxaliplatin response across the 21 CRC cell lines. These 58 genes were mainly enriched in small molecules biochemistry, Wnt/β-catenin signaling and EMT pathways. The latter two pathways were predicted to be activated in oxaliplatin-resistant CRC cell lines. Moreover, 15 genes were validated by qPCR that their expression levels were actually closely correlated to their response to oxaliplatin, in line with the biocomputation prediction. Taken together, our work might provide potential biomarkers for oxaliplatin sensitivity in CRC cell lines and therapeutic targets for combinational therapy with oxaliplatin.

  19. Translocation by T7 RNA polymerase: a sensitively poised Brownian ratchet.

    PubMed

    Guo, Qing; Sousa, Rui

    2006-04-21

    Studies of halted T7 RNA polymerase (T7RNAP) elongation complexes (ECs) or of T7RNAP transcription against roadblocks due to DNA-bound proteins indicate that T7RNAP translocates via a passive Brownian ratchet mechanism. Crystal structures of T7RNAP ECs suggest that translocation involves an active power-stroke. However, neither solution studies of halted or slowed T7RNAP ECs, nor crystal structures of static complexes, are necessarily relevant to how T7RNAP translocates during rapid elongation. A recent single molecule study of actively elongating T7RNAPs provides support for the Brownian ratchet mechanism. Here, we obtain additional evidence for the existence of a Brownian ratchet during active T7RNAP elongation by showing that both rapidly elongating and halted complexes are equally sensitive to pyrophosphate. Using chemical nucleases tethered to the polymerase we achieve sub-ångström resolution in measuring the average position of halted T7RNAP ECs and find that the positional equilibrium of the EC is sensitively poised between pre-translocated and post-translocated states. This may be important in maximizing the sensitivity of the polymerase to sequences that cause pausing or termination. We also confirm that a crystallographically observed disorder to order transition in a loop formed by residues 589-612 also occurs in solution and is coupled to pyrophosphate or NTP release. This transition allows the loop to make interactions with the DNA that help stabilize the laterally mobile, ligand-free EC against dissociation.

  20. Sensitive Detection of Protein and miRNA Cancer Biomarkers using Silicon-Based Photonic Crystals and A Resonance Coupling Laser Scanning Platform

    PubMed Central

    George, Sherine; Chaudhery, Vikram; Lu, Meng; Takagi, Miki; Amro, Nabil; Pokhriyal, Anusha; Tan, Yafang; Ferreira, Placid; Cunningham, Brian T.

    2013-01-01

    Enhancement of the fluorescent output of surface-based fluorescence assays by performing them upon nanostructured photonic crystal (PC) surfaces has been demonstrated to increase signal intensities by >8000×. Using the multiplicative effects of optical resonant coupling to the PC in increasing the electric field intensity experienced by fluorescent labels (“enhanced excitation”) and the spatially biased funneling of fluorophore emissions through coupling to PC resonances (“enhanced extraction”), PC enhanced fluorescence (PCEF) can be adapted to reduce the limits of detection of disease biomarker assays, and to reduce the size and cost of high sensitivity detection instrumentation. In this work, we demonstrate the first silicon-based PCEF detection platform for multiplexed biomarker assay. The sensor in this platform is a silicon-based PC structure, comprised of a SiO2 grating that is overcoated with a thin film of high refractive index TiO2 and is produced in a semiconductor foundry for low cost, uniform, and reproducible manufacturing. The compact detection instrument that completes this platform was designed to efficiently couples fluorescence excitation from a semiconductor laser to the resonant optical modes of the PC, resulting in elevated electric field strength that is highly concentrated within the region <100 nm from the PC surface. This instrument utilizes a cylindrically focused line to scan a microarray in <1 minute. To demonstrate the capabilities of this sensor-detector platform, microspot fluorescent sandwich immunoassays using secondary antibodies labeled with Cy5 for two cancer biomarkers (TNF-α and IL-3) were performed. Biomarkers were detected at concentrations as low as 0.1 pM. In a fluorescent microarray for detection of a breast cancer miRNA biomarker miR-21, the miRNA was detectable at a concentration of 0.6 pM. PMID:23963502

  1. pH-Sensitive carboxymethyl chitosan-modified cationic liposomes for sorafenib and siRNA co-delivery.

    PubMed

    Yao, Yao; Su, Zhihui; Liang, Yanchao; Zhang, Na

    2015-01-01

    Combination of chemotherapeutic drug and small interfering RNA (siRNA) can affect multiple disease pathways and has been proven effective in suppressing tumor progression. Co-delivery of drug and siRNA within a same nanocarrier is a vital means in this field. The present study aimed at the development of a pH-sensitive liposome to co-deliver drug and siRNA to tumor region. Driven by the electrostatic interaction, the pH-sensitive material, carboxymethyl chitosan (CMCS), was coated onto the surface of the cationic liposome (CL) preloaded with sorafenib (Sf) and siRNA (Si). To evaluate whether the resulting CMCS-modified Sf and siRNA co-delivery cationic liposome (CMCS-SiSf-CL) enhanced antitumor efficiency after systematic administration, in vitro and in vivo experiments were evaluated in HepG2 cells and the H22 cells-bearing Kunming mice model. The experimental results demonstrated that CMCS-SiSf-CL was able to condense siRNA efficiently and protect siRNA from being degraded by serum and RNase. The release rate of Sf from CMCS-modified liposome exhibited pH-sensitive release behavior. Furthermore, in vitro cellular uptake results showed that CMCS-SiSf-CL yielded higher fluorescence intensity at pH 6.5 than at pH 7.4, and that siRNA could be delivered to tumor site by CMCS-SiSf-CL in vivo. The in vivo antitumor efficacy showed that CMCS-Sf-CL inhibits tumor growth effectively when compared with free Sf solution. In current experimental conditions, this liposomal formulation did not show significant toxicity both in vitro and in vivo. Therefore, co-delivering Sf with siRNA by CMCS-SiSf-CL might provide a promising approach for tumor therapy. PMID:26491291

  2. pH-Sensitive carboxymethyl chitosan-modified cationic liposomes for sorafenib and siRNA co-delivery

    PubMed Central

    Yao, Yao; Su, Zhihui; Liang, Yanchao; Zhang, Na

    2015-01-01

    Combination of chemotherapeutic drug and small interfering RNA (siRNA) can affect multiple disease pathways and has been proven effective in suppressing tumor progression. Co-delivery of drug and siRNA within a same nanocarrier is a vital means in this field. The present study aimed at the development of a pH-sensitive liposome to co-deliver drug and siRNA to tumor region. Driven by the electrostatic interaction, the pH-sensitive material, carboxymethyl chitosan (CMCS), was coated onto the surface of the cationic liposome (CL) preloaded with sorafenib (Sf) and siRNA (Si). To evaluate whether the resulting CMCS-modified Sf and siRNA co-delivery cationic liposome (CMCS-SiSf-CL) enhanced antitumor efficiency after systematic administration, in vitro and in vivo experiments were evaluated in HepG2 cells and the H22 cells-bearing Kunming mice model. The experimental results demonstrated that CMCS-SiSf-CL was able to condense siRNA efficiently and protect siRNA from being degraded by serum and RNase. The release rate of Sf from CMCS-modified liposome exhibited pH-sensitive release behavior. Furthermore, in vitro cellular uptake results showed that CMCS-SiSf-CL yielded higher fluorescence intensity at pH 6.5 than at pH 7.4, and that siRNA could be delivered to tumor site by CMCS-SiSf-CL in vivo. The in vivo antitumor efficacy showed that CMCS-Sf-CL inhibits tumor growth effectively when compared with free Sf solution. In current experimental conditions, this liposomal formulation did not show significant toxicity both in vitro and in vivo. Therefore, co-delivering Sf with siRNA by CMCS-SiSf-CL might provide a promising approach for tumor therapy. PMID:26491291

  3. Exosomes derived from alcohol-treated hepatocytes horizontally transfer liver specific miRNA-122 and sensitize monocytes to LPS

    PubMed Central

    Momen-Heravi, Fatemeh; Bala, Shashi; Kodys, Karen; Szabo, Gyongyi

    2015-01-01

    Hepatocyte damage and inflammation in monocytes/macrophages are central to the pathogenesis of alcoholic hepatitis (AH). MicroRNAs (miRNAs) regulate all of these processes. MiRNA-122 is abundantly expressed in hepatocytes while monocytes/macrophages have low levels. The role of exosomes in AH and possible cross talk between hepatocyte-derived exosomes and immune cells is not explored yet. Here, we show that the number of exosomes significantly increases in the sera of healthy individuals after alcohol binge drinking and in mice after binge or chronic alcohol consumption. Exosomes isolated from sera after alcohol consumption or from in vitro ethanol-treated hepatocytes contained miRNA-122. Exosomes derived from ethanol-treated Huh7.5 cells were taken up by the recipients THP1 monocytes and horizontally transferred a mature form of liver-specific miRNA-122. In vivo, liver mononuclear cells and Kupffer cells from alcohol-fed mice had increased miRNA-122 levels. In monocytes, miRNA-122 transferred via exosomes inhibited the HO-1 pathway and sensitized to LPS stimulation and increased levels of pro-inflammatory cytokines. Finally, inflammatory effects of exosomes from ethanol-treated hepatocytes were prevented by using RNA interference via exosome-mediated delivery of a miRNA-122 inhibitor. These results demonstrate that first, exosomes mediate communication between hepatocytes and monocytes/macrophages and second, hepatocyte-derived miRNA-122 can reprogram monocytes inducing sensitization to LPS. PMID:25973575

  4. Sensitivity of Carrot Cell Cultures and RNA Polymerase II to Amatoxins 1

    PubMed Central

    Little, Michael C.; Preston, James F.

    1985-01-01

    Protoplast and cell suspension cultures of Daucus carota L. were evaluated for their sensitivity toward the three amatoxin derivatives, α-amanitin, 6′-deoxy-α-amanitin, and 6′-O-methyl-α-amanitin using inhibition of DNA synthesis to measure cell viability. Protoplasts appeared approximately 10-fold more refractory than suspension cells and α-amanitin was much less effective than the other two amatoxins, even though Ki values for isolated RNA polymerase II were similar (4-5 nanomolar). Additional studies evaluating the recoveries of all three amatoxins from cell suspension supernates indicate one basis for these differences to be the selective degradation of α-amanitin. A mechanism involving the activation of the hydroxyindole moiety of the α-amanitin is thus invoked to explain these differences and we postulate the involvement of plant oxidases in this role. PMID:16664072

  5. Single molecule fluorescence microscopy for ultra-sensitive RNA expression profiling

    NASA Astrophysics Data System (ADS)

    Hesse, Jan; Jacak, Jaroslaw; Regl, Gerhard; Eichberger, Thomas; Aberger, Fritz; Schlapak, Robert; Howorka, Stefan; Muresan, Leila; Frischauf, Anna-Maria; Schütz, Gerhard J.

    2007-02-01

    We developed a microarray analysis platform for ultra-sensitive RNA expression profiling of minute samples. It utilizes a novel scanning system for single molecule fluorescence detection on cm2 size samples in combination with specialized biochips, optimized for low autofluorescence and weak unspecific adsorption. 20 μg total RNA was extracted from 10 6 cells of a human keratinocyte cell line (HaCaT) and reversely transcribed in the presence of Alexa647-aha-dUTP. 1% of the resulting labeled cDNA was used for complex hybridization to a custom-made oligonucleotide microarray representing a set of 125 different genes. For low abundant genes, individual cDNA molecules hybridized to the microarray spots could be resolved. Single cDNA molecules hybridized to the chip surface appeared as diffraction limited features in the fluorescence images. The à trous wavelet method was utilized for localization and counting of the separated cDNA signals. Subsequently, the degree of labeling of the localized cDNA molecules was determined by brightness analysis for the different genes. Variations by factors up to 6 were found, which in conventional microarray analysis would result in a misrepresentation of the relative abundance of mRNAs.

  6. CCAT1 is an enhancer-templated RNA that predicts BET sensitivity in colorectal cancer.

    PubMed

    McCleland, Mark L; Mesh, Kathryn; Lorenzana, Edward; Chopra, Vivek S; Segal, Ehud; Watanabe, Colin; Haley, Benjamin; Mayba, Oleg; Yaylaoglu, Murat; Gnad, Florian; Firestein, Ron

    2016-02-01

    Colon tumors arise in a stepwise fashion from either discrete genetic perturbations or epigenetic dysregulation. To uncover the key epigenetic regulators that drive colon cancer growth, we used a CRISPR loss-of-function screen and identified a number of essential genes, including the bromodomain and extraterminal (BET) protein BRD4. We found that BRD4 is critical for colon cancer proliferation, and its knockdown led to differentiation effects in vivo. JQ1, a BET inhibitor, preferentially reduced growth in a subset of epigenetically dysregulated colon cancers characterized by the CpG island methylator phenotype (CIMP). Integrated transcriptomic and genomic analyses defined a distinct superenhancer in CIMP+ colon cancers that regulates cMYC transcription. We found that the long noncoding RNA colon cancer-associated transcript 1 (CCAT1) is transcribed from this superenhancer and is exquisitely sensitive to BET inhibition. Concordantly, cMYC transcription and cell growth were tightly correlated with the presence of CCAT1 RNA in a variety of tumor types. Taken together, we propose that CCAT1 is a clinically tractable biomarker for identifying patients who are likely to benefit from BET inhibitors. PMID:26752646

  7. Isolation and characterization of temperature-sensitive RNA polymerase II mutants of Saccharomyces cerevisiae.

    PubMed Central

    Himmelfarb, H J; Simpson, E M; Friesen, J D

    1987-01-01

    Three independent, recessive, temperature-sensitive (Ts-) conditional lethal mutations in the largest subunit of Saccharomyces cerevisiae RNA polymerase II (RNAP II) have been isolated after replacement of a portion of the wild-type gene (RPO21) by a mutagenized fragment of the cloned gene. Measurements of cell growth, viability, and total RNA and protein synthesis showed that rpo21-1, rpo21-2, and rpo21-3 mutations caused a slow shutoff of RNAP II activity in cells shifted to the nonpermissive temperature (39 degrees C). Each mutant displayed a distinct phenotype, and one of the mutant enzymes (rpo21-1) was completely deficient in RNAP II activity in vitro. RNAP I and RNAP III in vitro activities were not affected. These results were consistent with the notion that the genetic lesions affect RNAP II assembly or holoenzyme stability. DNA sequencing revealed that in each case the mutations involved nonconservative amino acid substitutions, resulting in charge changes. The lesions harbored by all three rpo21 Ts- alleles lie in DNA sequence domains that are highly conserved among genes that encode the largest subunits of RNAP from a variety of eucaryotes; one mutation lies in a possible Zn2+ binding domain. PMID:3299061

  8. Aptamer-miRNA-212 Conjugate Sensitizes NSCLC Cells to TRAIL

    PubMed Central

    Iaboni, Margherita; Russo, Valentina; Fontanella, Raffaela; Roscigno, Giuseppina; Fiore, Danilo; Donnarumma, Elvira; Esposito, Carla Lucia; Quintavalle, Cristina; Giangrande, Paloma H; de Franciscis, Vittorio; Condorelli, Gerolama

    2016-01-01

    TNF-related apoptosis-inducing ligand (TRAIL) is a promising antitumor agent for its remarkable ability to selectively induce apoptosis in cancer cells, without affecting the viability of healthy bystander cells. The TRAIL tumor suppressor pathway is deregulated in many human malignancies including lung cancer. In human non-small cell lung cancer (NSCLC) cells, sensitization to TRAIL therapy can be restored by increasing the expression levels of the tumor suppressor microRNA-212 (miR-212) leading to inhibition of the anti-apoptotic protein PED/PEA-15 implicated in treatment resistance. In this study, we exploited a previously described RNA aptamer inhibitor of the tyrosine kinase receptor Axl (GL21.T) expressed on lung cancer cells, as a means to deliver miR-212 into human NSCLC cells expressing Axl. We demonstrate efficient delivery of miR-212 following conjugation of the miR to GL21.T (GL21.T-miR212 chimera). We show that the chimera downregulates PED and restores TRAIL-mediate cytotoxicity in cancer cells. Importantly, treatment of Axl+ lung cancer cells with the chimera resulted in (i) an increase in caspase activation and (ii) a reduction of cell viability in combination with TRAIL therapy. In conclusion, we demonstrate that the GL21.T-miR212 chimera can be employed as an adjuvant to TRAIL therapy for the treatment of lung cancer. PMID:27111415

  9. Use of magnetic beads versus guanidium thiocyanate-phenol-chloroform RNA extraction followed by polymerase chain reaction for the rapid, sensitive detection of enterovirus RNA.

    PubMed

    Beaulieux, F; See, D M; Leparc-Goffart, I; Aymard, M; Lina, B

    1997-01-01

    The current study compares the sensitivity of RNA extraction using magnetic beads versus that of a standard extraction method. Streptavadin-coated magnetic beads were labelled with a biotinylated, enterovirus-specific oligonucleotide. RNA was extracted using labelled beads or guanidium thiocyanate-phenol-chloroform from 1, 0.1 and 0.01 TCID50/100 microliters of stock coxsackievirus types A9 and B3, echovirus type 11, enterovirus type 70 and poliovirus type 1. Each strain was tested three times. RNA extraction using magnetic beads was > 50% faster than the standard method. The RNA was amplified using RT-PCR, and the products were detected using agarose gel electrophoresis; 6/15 and 7/15 samples at an initial concentration of 0.01 TCID50/100 microliters were detected using magnetic beads or standard extraction, respectively. Negative-stain electron microscopy was used to determine that 0.01 TCID50/100 microliters of coxsackievirus B3 contained approximately 3 genomes. Thus, use of magnetic beads labelled with an enterovirus-specific oligonucleotide was less toxic, more rapid and as sensitive as the current standard RNA extraction method.

  10. Label-free fluorescence strategy for sensitive microRNA detection based on isothermal exponential amplification and graphene oxide.

    PubMed

    Li, Wei; Hou, Ting; Wu, Min; Li, Feng

    2016-02-01

    MicroRNAs (miRNAs) play an important role in many biological processes, and have been regarded as potential targets and biomarkers in cancer diagnosis and therapy. Also, to meet the big challenge imposed by the characteristics of miRNAs, such as small size and vulnerability to enzymatic digestion, it is of great importance to develop accurate, sensitive and simple miRNA assays. Herein, we developed a label-free fluorescence strategy for sensitive miRNA detection by combining isothermal exponential amplification and the unique features of SYBR Green I (SG) and graphene oxide (GO), in which SG gives significantly enhanced fluorescence upon intercalation into double-stranded DNAs (dsDNAs), and GO selectively adsorbs miRNA, single-stranded DNA and SG, to protect miRNA from enzymatic digestion, and to quench the fluorescence of the adsorbed SG. In the presence of the target miRNA, the ingeniously designed hairpin probe (HP) is unfolded and the subsequent polymerization and strand displacement reaction takes place to initiate the target recycling process. The newly formed dsDNAs are then recognized and cleaved by the nicking enzyme, generating new DNA triggers with the same sequence as the target miRNA, which hybridize with intact HPs to initiate new extension reactions. As a result, the circular exponential amplification for target miRNA is achieved and large amount of dsDNAs are formed to generate significantly enhanced fluorescence upon the intercalation of SG. Thus sensitive and selective fluorescence miRNA detection is realized, and the detection limit of 3 fM is obtained. Besides, this method exhibits additional advantages of simplicity and low cost, since expensive and tedious labeling process is avoided. Therefore, the as-proposed label-free fluorescence strategy has great potential in the applications in miRNA-related clinical practices and biochemical researches. PMID:26653431

  11. Label-free fluorescence strategy for sensitive microRNA detection based on isothermal exponential amplification and graphene oxide.

    PubMed

    Li, Wei; Hou, Ting; Wu, Min; Li, Feng

    2016-01-01

    MicroRNAs (miRNAs) play an important role in many biological processes, and have been regarded as potential targets and biomarkers in cancer diagnosis and therapy. Also, to meet the big challenge imposed by the characteristics of miRNAs, such as small size and vulnerability to enzymatic digestion, it is of great importance to develop accurate, sensitive and simple miRNA assays. Herein, we developed a label-free fluorescence strategy for sensitive miRNA detection by combining isothermal exponential amplification and the unique features of SYBR Green I (SG) and graphene oxide (GO), in which SG gives significantly enhanced fluorescence upon intercalation into double-stranded DNAs (dsDNAs), and GO selectively adsorbs miRNA, single-stranded DNA and SG, to protect miRNA from enzymatic digestion, and to quench the fluorescence of the adsorbed SG. In the presence of the target miRNA, the ingeniously designed hairpin probe (HP) is unfolded and the subsequent polymerization and strand displacement reaction takes place to initiate the target recycling process. The newly formed dsDNAs are then recognized and cleaved by the nicking enzyme, generating new DNA triggers with the same sequence as the target miRNA, which hybridize with intact HPs to initiate new extension reactions. As a result, the circular exponential amplification for target miRNA is achieved and large amount of dsDNAs are formed to generate significantly enhanced fluorescence upon the intercalation of SG. Thus sensitive and selective fluorescence miRNA detection is realized, and the detection limit of 3 fM is obtained. Besides, this method exhibits additional advantages of simplicity and low cost, since expensive and tedious labeling process is avoided. Therefore, the as-proposed label-free fluorescence strategy has great potential in the applications in miRNA-related clinical practices and biochemical researches.

  12. tRNA Modifying Enzymes, NSUN2 and METTL1, Determine Sensitivity to 5-Fluorouracil in HeLa Cells

    PubMed Central

    Hori, Masato; Okada, Kaoru; Yazama, Futoshi; Konishi, Hiroaki; Xiao, Yegui; Qi, Guangying; Shimamoto, Fumio; Ota, Takahide; Temme, Achim; Tatsuka, Masaaki

    2014-01-01

    Nonessential tRNA modifications by methyltransferases are evolutionarily conserved and have been reported to stabilize mature tRNA molecules and prevent rapid tRNA decay (RTD). The tRNA modifying enzymes, NSUN2 and METTL1, are mammalian orthologs of yeast Trm4 and Trm8, which are required for protecting tRNA against RTD. A simultaneous overexpression of NSUN2 and METTL1 is widely observed among human cancers suggesting that targeting of both proteins provides a novel powerful strategy for cancer chemotherapy. Here, we show that combined knockdown of NSUN2 and METTL1 in HeLa cells drastically potentiate sensitivity of cells to 5-fluorouracil (5-FU) whereas heat stress of cells revealed no effects. Since NSUN2 and METTL1 are phosphorylated by Aurora-B and Akt, respectively, and their tRNA modifying activities are suppressed by phosphorylation, overexpression of constitutively dephosphorylated forms of both methyltransferases is able to suppress 5-FU sensitivity. Thus, NSUN2 and METTL1 are implicated in 5-FU sensitivity in HeLa cells. Interfering with methylation of tRNAs might provide a promising rationale to improve 5-FU chemotherapy of cancer. PMID:25233213

  13. A first-generation multiplex biomarker analysis of urine for the early detection of prostate cancer.

    PubMed

    Laxman, Bharathi; Morris, David S; Yu, Jianjun; Siddiqui, Javed; Cao, Jie; Mehra, Rohit; Lonigro, Robert J; Tsodikov, Alex; Wei, John T; Tomlins, Scott A; Chinnaiyan, Arul M

    2008-02-01

    Although prostate-specific antigen (PSA) serum level is currently the standard of care for prostate cancer screening in the United States, it lacks ideal specificity and additional biomarkers are needed to supplement or potentially replace serum PSA testing. Emerging evidence suggests that monitoring the noncoding RNA transcript PCA3 in urine may be useful in detecting prostate cancer in patients with elevated PSA levels. Here, we show that a multiplex panel of urine transcripts outperforms PCA3 transcript alone for the detection of prostate cancer. We measured the expression of seven putative prostate cancer biomarkers, including PCA3, in sedimented urine using quantitative PCR on a cohort of 234 patients presenting for biopsy or radical prostatectomy. By univariate analysis, we found that increased GOLPH2, SPINK1, and PCA3 transcript expression and TMPRSS2:ERG fusion status were significant predictors of prostate cancer. Multivariate regression analysis showed that a multiplexed model, including these biomarkers, outperformed serum PSA or PCA3 alone in detecting prostate cancer. The area under the receiver-operating characteristic curve was 0.758 for the multiplexed model versus 0.662 for PCA3 alone (P = 0.003). The sensitivity and specificity for the multiplexed model were 65.9% and 76.0%, respectively, and the positive and negative predictive values were 79.8% and 60.8%, respectively. Taken together, these results provide the framework for the development of highly optimized, multiplex urine biomarker tests for more accurate detection of prostate cancer.

  14. RNA aptamer based electrochemical biosensor for sensitive and selective detection of cAMP.

    PubMed

    Zhao, Fulin; Xie, Qingyun; Xu, Mingfei; Wang, Shouyu; Zhou, Jiyong; Liu, Fei

    2015-04-15

    Cyclic adenosine monophosphate (cAMP) is an important small biological molecule associated with the healthy state of living organism. In order to realize highly sensitive and specific detection of cAMP, here an RNA aptamer and electrochemical impedance spectroscopy (EIS) based biosensor enhanced by gold nanoparticles electrodeposited on the surface of gold electrode is designed. The designed aptasensor has a wide effective measuring range from 50pM to 250pM with a detection limit of 50pM in PBS buffer, and an effective measuring range from 50nM to 1μM with a detection limit of 50nM in serum. The designed biosensor is also able to detect cAMP with high sensitivity, specificity, and stability. Since the biosensor can be easily fabricated with low cost and repeatedly used for at least two times, it owns great potential in wide application fields such as clinical test and food inspection, etc.

  15. N-ethylmaleimide-sensitive factor siRNA inhibits the release of Weibel-Palade bodies in endothelial cells

    PubMed Central

    Zhou, Yong; Yang, Shui-Xiang; Yue, Yu-Nan; Wei, Xiao-Fei; Liu, Yan

    2016-01-01

    The aim of the present study was to examine the effect of small interfering RNA (siRNA) methods on the expression of N-ethylmaleimide sensitive factor (NSF) and Weibel-Palade body (WPB) release in endothelial cells. A small hairpin RNA (shRNA), mediated with an adenovirus vector, was designed to target the N-terminal functional area of NSF. Subsequently, viruses were transfected into human aortic endothelial cells. The mRNA and protein expression levels of NSF were detected using reverse transcription-quantitative polymerase chain reaction and Western blot analyses, respectively, and the release of WPBs in the endothelial cells was examined using immunofluorescence. The mRNA expression of NSF in the endothelial cells, which were transfected with the adenoviruses carrying the NSF-shRNA was significantly decreased, compared with the negative control group (P=0.035) and blank control group (P=0.02). In addition, the mRNA expression of NSF was gradually decreased as duration increased; there were marked differences between the 24, 48 and 72 h groups (P<0.05). The protein expression of NSF was significantly decreased in the experimental group, compared with the negative control group (P=0.004) and blank control group (P=0.031), however, no difference was observed between the negative control and blank control groups (P=0.249). The immunofluorescence staining showed that the release of WPBs in the endothelial cells induced with thrombin was inhibited markedly following transfection with the virus carrying the NSF-shRNA. Therefore NSF-siRNA inhibited the mRNA and protein expression levels of NSF, and inhibited the release of WPBs in endothelial cells induced with thrombin. These results suggested that NSF-siRNA may be valuable for preventing and treating atherosclerosis and acute coronary syndrome. PMID:27277949

  16. Sensitive RNA detection by combining three-way junction formation and primer generation-rolling circle amplification.

    PubMed

    Murakami, Taku; Sumaoka, Jun; Komiyama, Makoto

    2012-02-01

    Recently, we developed a simple isothermal nucleic acid amplification reaction, primer generation-rolling circle amplification (PG-RCA), to detect specific DNA sequences with great sensitivity and large dynamic range. In this paper, we combined PG-RCA with a three-way junction (3WJ) formation, and detected specific RNA molecules with high sensitivity and specificity in a one-step and isothermal reaction format. In the presence of target RNA, 3WJ probes (primer and template) are designed to form a 3WJ structure, from which multiple signal primers for the following PG-RCA can be generated by repeating primer extension, nicking and signal primer dissociation. Although this signal primer generation is a linear amplification process, the PG-RCA exponentially can amplify these signal primers and thus even a very small amount of RNA specimen can be detected. After optimizing the structures of 3WJ probes, the detection limit of this assay was 15.9 zmol (9.55 × 10(3) molecules) of synthetic RNA or 143 zmol (8.6 × 10(4) molecules) of in vitro transcribed human CD4 mRNA. Further, the applicability of this assay to detect CD4 mRNA in a human mRNA sample was demonstrated.

  17. Multiplexed Simultaneous High Sensitivity Sensors with High-Order Mode Based on the Integration of Photonic Crystal 1 × 3 Beam Splitter and Three Different Single-Slot PCNCs.

    PubMed

    Zhou, Jian; Huang, Lijun; Fu, Zhongyuan; Sun, Fujun; Tian, Huiping

    2016-01-01

    We simulated an efficient method for the sensor array of high-sensitivity single-slot photonic crystal nanobeam cavities (PCNCs) on a silicon platform. With the combination of a well-designed photonic crystal waveguide (PhCW) filter and an elaborate single-slot PCNC, a specific high-order resonant mode was filtered for sensing. A 1 × 3 beam splitter carefully established was implemented to split channels and integrate three sensors to realize microarrays. By applying the three-dimensional finite-difference-time-domain (3D-FDTD) method, the sensitivities calculated were S₁ = 492 nm/RIU, S₂ = 244 nm/RIU, and S₃ = 552 nm/RIU, respectively. To the best of our knowledge, this is the first multiplexing design in which each sensor cite features such a high sensitivity simultaneously. PMID:27399712

  18. Multiplexed Simultaneous High Sensitivity Sensors with High-Order Mode Based on the Integration of Photonic Crystal 1 × 3 Beam Splitter and Three Different Single-Slot PCNCs.

    PubMed

    Zhou, Jian; Huang, Lijun; Fu, Zhongyuan; Sun, Fujun; Tian, Huiping

    2016-07-07

    We simulated an efficient method for the sensor array of high-sensitivity single-slot photonic crystal nanobeam cavities (PCNCs) on a silicon platform. With the combination of a well-designed photonic crystal waveguide (PhCW) filter and an elaborate single-slot PCNC, a specific high-order resonant mode was filtered for sensing. A 1 × 3 beam splitter carefully established was implemented to split channels and integrate three sensors to realize microarrays. By applying the three-dimensional finite-difference-time-domain (3D-FDTD) method, the sensitivities calculated were S₁ = 492 nm/RIU, S₂ = 244 nm/RIU, and S₃ = 552 nm/RIU, respectively. To the best of our knowledge, this is the first multiplexing design in which each sensor cite features such a high sensitivity simultaneously.

  19. Multiplexed Simultaneous High Sensitivity Sensors with High-Order Mode Based on the Integration of Photonic Crystal 1 × 3 Beam Splitter and Three Different Single-Slot PCNCs

    PubMed Central

    Zhou, Jian; Huang, Lijun; Fu, Zhongyuan; Sun, Fujun; Tian, Huiping

    2016-01-01

    We simulated an efficient method for the sensor array of high-sensitivity single-slot photonic crystal nanobeam cavities (PCNCs) on a silicon platform. With the combination of a well-designed photonic crystal waveguide (PhCW) filter and an elaborate single-slot PCNC, a specific high-order resonant mode was filtered for sensing. A 1 × 3 beam splitter carefully established was implemented to split channels and integrate three sensors to realize microarrays. By applying the three-dimensional finite-difference-time-domain (3D-FDTD) method, the sensitivities calculated were S1 = 492 nm/RIU, S2 = 244 nm/RIU, and S3 = 552 nm/RIU, respectively. To the best of our knowledge, this is the first multiplexing design in which each sensor cite features such a high sensitivity simultaneously. PMID:27399712

  20. Low-cost, multiplexed biosensor for disease diagnosis

    NASA Astrophysics Data System (ADS)

    Myatt, Christopher J.; Delaney, Marie; Todorof, Kathryn; Heil, James; Givens, Monique; Schooley, Robert T.; Lochhead, Michael J.

    2009-02-01

    Cost-effective disease diagnosis in resource-limited settings remains a critical global health challenge. Qualitative rapid tests based on lateral flow technology provide valuable screening information, but require relatively expensive confirmatory tests and generally lack quantitation. We report on a fluorescence technology that combines low cost instrumented readout with passive pumping in a disposable cartridge. The detection system utilizes a novel waveguide illumination approach in conjunction with commercial CMOS imagers. Total instrument cost in production are projected to be around $100 This cost structure and instrument ease of use will enable use in point-of-care settings, outside of centralized laboratories. The system has been used for detection and analysis of proteins, antibodies, nucleic acids, and cells. Here we will report first on our development of a multiplexed, array-based serology assay for HIV and common AIDS co-infections. Data will be presented for HIV/HCV antibody testing in human serum samples. In addition, we will present data on the use of the system for sensitive detection of bacterial RNA. Current detection limit for the model multiplexed RNA sandwich assay is 1 femtomolar target RNA. Finally, a high magnification version of the system is used to image immunostained human T cells.

  1. Fidelity and enhanced sensitivity of differential transcription profiles following linear amplification of nanogram amounts of endothelial mRNA

    NASA Technical Reports Server (NTRS)

    Polacek, Denise C.; Passerini, Anthony G.; Shi, Congzhu; Francesco, Nadeene M.; Manduchi, Elisabetta; Grant, Gregory R.; Powell, Steven; Bischof, Helen; Winkler, Hans; Stoeckert, Christian J Jr; Davies, Peter F.

    2003-01-01

    Although mRNA amplification is necessary for microarray analyses from limited amounts of cells and tissues, the accuracy of transcription profiles following amplification has not been well characterized. We tested the fidelity of differential gene expression following linear amplification by T7-mediated transcription in a well-established in vitro model of cytokine [tumor necrosis factor alpha (TNFalpha)]-stimulated human endothelial cells using filter arrays of 13,824 human cDNAs. Transcriptional profiles generated from amplified antisense RNA (aRNA) (from 100 ng total RNA, approximately 1 ng mRNA) were compared with profiles generated from unamplified RNA originating from the same homogeneous pool. Amplification accurately identified TNFalpha-induced differential expression in 94% of the genes detected using unamplified samples. Furthermore, an additional 1,150 genes were identified as putatively differentially expressed using amplified RNA which remained undetected using unamplified RNA. Of genes sampled from this set, 67% were validated by quantitative real-time PCR as truly differentially expressed. Thus, in addition to demonstrating fidelity in gene expression relative to unamplified samples, linear amplification results in improved sensitivity of detection and enhances the discovery potential of high-throughput screening by microarrays.

  2. Sensitivity of Ribosomal RNA Character Sampling in the Phylogeny of Rhabditida

    PubMed Central

    Holovachov, Oleksandr; Camp, Lauren; Nadler, Steven A.

    2015-01-01

    Near-full-length 18S and 28S rRNA gene sequences were obtained for 33 nematode species. Datasets were constructed based on secondary structure and progressive multiple alignments, and clades were compared for phylogenies inferred by Bayesian and maximum likelihood methods. Clade comparisons were also made following removal of ambiguously aligned sites as determined using the program ProAlign. Different alignments of these data produced tree topologies that differed, sometimes markedly, when analyzed by the same inference method. With one exception, the same alignment produced an identical tree topology when analyzed by different methods. Removal of ambiguously aligned sites altered the tree topology and also reduced resolution. Nematode clades were sensitive to differences in multiple alignments, and more than doubling the amount of sequence data by addition of 28S rRNA did not fully mitigate this result. Although some individual clades showed substantially higher support when 28S data were combined with 18S data, the combined analysis yielded no statistically significant increases in the number of clades receiving higher support when compared to the 18S data alone. Secondary structure alignment increased accuracy in positional homology assignment and, when used in combination with paired-site substitution models, these structural hypotheses of characters and improved models of character state change yielded high levels of phylogenetic resolution. Phylogenetic results included strong support for inclusion of Daubaylia potomaca within Cephalobidae, whereas the position of Fescia grossa within Tylenchina varied depending on the alignment, and the relationships among Rhabditidae, Diplogastridae, and Bunonematidae were not resolved. PMID:26941463

  3. Differences in correlation of mRNA gene expression in mice sensitive and resistant to radiation-induced pulmonary fibrosis

    SciTech Connect

    Johnston, C.J.; Piedboeuf, B.; Finkelstein, J.N.; Baggs, R.; Rubin, P.

    1995-05-01

    Fibrosis, characterized by the accumulation of collagen, is a late result of thoracic irradiation. The purpose of this study was to determine if extracellular matrix protein and transforming growth factor {beta} mRNA expression are altered late in the course of pulmonary fibrosis after irradiation, and then to determine if these changes differ between two strains of mice which vary in their sensitivity to radiation. Radiation-sensitive (C57BL/6) and radiation-resistant (C3H/HeJ) mice were irradiated with a single dose of 5 or 12.5 Gy to the thorax. Total lung RNA was prepared and immobilized by Northern and slot blotting and hybridized with radiolabeled cDNA probes for collagens I, III and IV, fibronectin, and transforming growth factor {beta}{sub 1} and {beta}{sub 3}. Autoradiographic data were quantified by video densitometry and results normalized to a control probe encoding for glyceralde-hyde-3-phosphate dehydrogenase. Alterations in mRNA abundance were observed in the sensitive mice at all times, while levels in the resistant mice were unaffected until 26 weeks after irradiation. The relationship between extracellular matrix protein per se and increased mRNA abundance suggests that late matrix protein accumulation may be a function of gene expression. Differences in levels of transforming growth factor {beta}mRNA may lead to strain-dependent variation in fibrotic response and may also contribute to the radiation-induced component of pulmonary fibrosis. 32 refs., 5 figs.

  4. Gold Nanoparticle Coated Silica Nanorods for Sensitive Visual Detection of microRNA on a Lateral Flow Strip Biosensor.

    PubMed

    Takalkar, Sunitha; Xu, Hui; Chen, Jiao; Baryeh, Kwaku; Qiu, Wanwei; Zhao, Julia X; Liu, And Guodong

    2016-01-01

    We present a rapid and highly sensitive approach for visual detection of microRNA (miRNA) using a gold nanoparticles coated silica nanorod label and lateral flow strip biosensor. Gold nanoparticles were decorated on the silica nanorod surface by a seeding and growth procedure. A single strand DNA probe was immobilized on the gold nanoparticles-silica nanorod surface by a self-assembling process, and the formed DNA-gold nanoparticles-silica nanorod conjugate was used to construct the lateral flow nucleic acid biosensor for detecting miRNA. The captured gold nanoparticles-silica nanorods by sandwich-type hybridization reactions (DNA-RNA-DNA) on the test zone of the lateral flow nucleic acid biosensor produced the characteristic color bands, enabling visual detection of miRNA. After systematic optimization, the new lateral flow nucleic acid biosensor was capable of detecting 10 pM of the miRNA target without instrumentation, which is six times lower than that obtained with the gold nanoparticle-based lateral flow nucleic acid biosensor. The gold nanoparticles coated silica nanorod thus provides a new and sensitive nanolabel for visual detection of biological molecules on the lateral flow biosensor. PMID:27302581

  5. Pulmonary Codelivery of Doxorubicin and siRNA by pH-Sensitive Nanoparticles for Therapy of Metastatic Lung Cancer.

    PubMed

    Xu, Caina; Wang, Ping; Zhang, Jingpeng; Tian, Huayu; Park, Kinam; Chen, Xuesi

    2015-09-01

    A pulmonary codelivery system that can simultaneously deliver doxorubicin (DOX) and Bcl2 siRNA to the lungs provides a promising local treatment strategy for lung cancers. In this study, DOX is conjugated onto polyethylenimine (PEI) by using cis-aconitic anhydride (CA, a pH-sensitive linker) to obtain PEI-CA-DOX conjugates. The PEI-CA-DOX/siRNA complex nanoparticles are formed spontaneously via electrostatic interaction between cationic PEI-CA-DOX and anionic siRNA. The drug release experiment shows that DOX releases faster at acidic pH than at pH 7.4. Moreover, PEI-CA-DOX/Bcl2 siRNA complex nanoparticles show higher cytotoxicity and apoptosis induction in B16F10 cells than those treated with either DOX or Bcl2 siRNA alone. When the codelivery systems are directly sprayed into the lungs of B16F10 melanoma-bearing mice, the PEI-CA-DOX/Bcl2 siRNA complex nanoparticles exhibit enhanced antitumor efficacy compared with the single delivery of DOX or Bcl2 siRNA. Compared with systemic delivery, most drug and siRNA show a long-term retention in the lungs via pulmonary delivery, and a considerable number of the drug and siRNA accumulate in tumor tissues of lungs, but rarely in normal lung tissues. The PEI-CA-DOX/Bcl2 siRNA complex nanoparticles are promising for the treatment of metastatic lung cancer by pulmonary delivery with low side effects on the normal tissues.

  6. Plasma miRNA levels correlate with sensitivity to bone mineral density in postmenopausal osteoporosis patients.

    PubMed

    Li, Hongqiu; Wang, Zhe; Fu, Qin; Zhang, Jing

    2014-11-01

    In our study, we detect the levels of three micro-RNAs (miRNAs; miR-21, miR-133a and miR-146a) in the plasma of 120 Chinese postmenopausal women who were divided into three groups (normal, osteopenia and osteoporosis) according to the T-scores. Downregulation of miR-21, as well as upregulation of miR-133a, was validated in the plasma of osteoporosis and osteopenia patients versus the normal group. The difference in expression regarding the miR-146a level in plasma among the three groups was not significant (p > 0.01). The circulating miRNA expression levels and bone mineral density (BMD) were examined during a multiple correlation analysis as a dependent variable after adjusting for age, weight and height. We have demonstrated that specific miRNAs species are significantly changed in the plasma of osteoporosis and osteopenia patients and correlated with the BMD. Our study suggested a potential use of miR-21 and miR-133a as sensitive and plasma biomarkers for postmenopausal osteoporosis.

  7. A novel, non-radioactive eukaryotic in vitro transcription assay for sensitive quantification of RNA polymerase II activity

    PubMed Central

    2014-01-01

    Background Many studies of the eukaryotic transcription mechanism and its regulation rely on in vitro assays. Conventional RNA polymerase II transcription assays are based on radioactive labelling of the newly synthesized RNA. Due to the inefficient in vitro transcription, the detection of the RNA involving purification and gel electrophoresis is laborious and not always quantitative. Results Herein, we describe a new, non-radioactive, robust and reproducible eukaryotic in vitro transcription assay that has been established in our laboratory. Upon transcription, the newly synthesized RNA is directly detected and quantified using the QuantiGene assay. Alternatively, the RNA can be purified and a primer extension followed by PCR detection or qPCR quantification can be performed. When applied to assess the activity of RNA polymerase II inhibitors, this new method allowed an accurate estimation of their relative potency. Conclusions Our novel assay provides a non-radioactive alternative to a standard in vitro transcription assay that allows for sensitive detection and precise quantification of the newly transcribed, unlabelled RNA and is particularly useful for quantification of strong transcriptional inhibitors like α-amanitin. Moreover, the method can be easily adapted to quantify the reaction yield and the transcription efficiency of other eukaryotic in vitro systems, thus providing a complementary tool for the field of transcriptional research. PMID:24694320

  8. Portable Multiplex Pathogen Detector

    SciTech Connect

    Visuri, S; McBride, M T; Matthews, D; Rao, R

    2002-07-15

    Tumor marker concentrations in serum provide useful information regarding clinical stage and prognosis of cancer and can thus be used for presymptomatic diagnostic purposes. Currently, detection and identification of soluble analytes in biological fluids is conducted by methods including bioassays, ELISA, PCR, DNA chip or strip tests. While these technologies are generally sensitive and specific, they are time consuming, labor intensive and cannot be multiplexed. Our goal is to develop a simple, point-of-care, portable, liquid array-based immunoassay device capable of simultaneous detection of a variety of cancer markers. Here we describe the development of assays for the detection of Serum Prostate Specific Antigen, and Ovalbumin from a single sample. The multiplexed immunoassays utilize polystyrene microbeads. The beads are imbedded with precise ratios of red and orange fluorescent dyes yielding an array of 100 beads, each with a unique spectral address (Figure 1). Each bead can be coated with capture antibodies specific for a given antigen. After antigen capture, secondary antibodies sandwich the bound antigen and are indirectly labeled by the fluorescent reporter phycoerythrin (PE). Each optically encoded and fluorescently-labeled microbead is then individually interrogated. A red laser excites the dye molecules imbedded inside the bead and classifies the bead to its unique bead set, and a green laser quantifies the assay at the bead surface. This technology has been proven to be comparable to the ELISA in terms of sensitivity and specificity. We also describe the laser-based instrumentation used to acquire fluorescent bead images Following the assay, droplets of bead suspension containing a mixture of bead classes were deposited onto filters held in place by a disposable plexiglass device and the resultant arrays viewed under the fluorescent imaging setup. Using the appropriate filter sets to extract the necessary red, orange and green fluorescence from the

  9. Potential RNA Binding Proteins in Saccharomyces Cerevisiae Identified as Suppressors of Temperature-Sensitive Mutations in Npl3

    PubMed Central

    Henry, M.; Borland, C. Z.; Bossie, M.; Silver, P. A.

    1996-01-01

    The NPL3 gene of the yeast Saccharomyces cerevisiae encodes a protein with similarity to heterogeneous nuclear ribonucleoproteins (hnRNPs). Npl3p has been implicated in many nuclear-related events including RNA export, protein import, and rRNA processing. Several temperature-sensitive alleles of NPL3 have been isolated. We now report the sequence of these alleles. For one allele, npl3-1, four complementation groups of suppressors have been isolated. The cognate genes for the two recessive mutants were cloned. One of these is the previously known RNA15, which, like NPL3, also encodes a protein with similarity to the vertebrate hnRNP A/B protein family. The other suppressor corresponds to a newly defined gene we term HRP1, which also encodes a protein with similarity to the hnRNP A/B proteins of vertebrates. Mutations in HRP1 suppress all npl3 temperature-sensitive alleles but do not bypass an npl3 null allele. We show that HRP1 is essential for cell growth and that the corresponding protein is located in the nucleus. The discovery of two hnRNP homologues that can partially suppress the function of Np13p, also an RNA binding protein, will be discussed in terms of the possible roles for Npl3p in RNA metabolism. PMID:8770588

  10. Graphene fluorescence switch-based cooperative amplification: a sensitive and accurate method to detection microRNA.

    PubMed

    Liu, Haiyun; Li, Lu; Wang, Qian; Duan, Lili; Tang, Bo

    2014-06-01

    MicroRNAs (miRNAs) play significant roles in a diverse range of biological progress and have been regarded as biomarkers and therapeutic targets in cancer treatment. Sensitive and accurate detection of miRNAs is crucial for better understanding their roles in cancer cells and further validating their function in clinical diagnosis. Here, we developed a stable, sensitive, and specific miRNAs detection method on the basis of cooperative amplification combining with the graphene oxide (GO) fluorescence switch-based circular exponential amplification and the multimolecules labeling of SYBR Green I (SG). First, the target miRNA is adsorbed on the surface of GO, which can protect the miRNA from enzyme digest. Next, the miRNA hybridizes with a partial hairpin probe and then acts as a primer to initiate a strand displacement reaction to form a complete duplex. Finally, under the action of nicking enzyme, universal DNA fragments are released and used as triggers to initiate next reaction cycle, constituting a new circular exponential amplification. In the proposed strategy, a small amount of target miRNA can be converted to a large number of stable DNA triggers, leading to a remarkable amplification for the target. Moreover, compared with labeling with a 1:1 stoichiometric ratio, multimolecules binding of intercalating dye SG to double-stranded DNA (dsDNA) can induce significant enhancement of fluorescence signal and further improve the detection sensitivity. The extraordinary fluorescence quenching of GO used here guarantees the high signal-to-noise ratio. Due to the protection for target miRNA by GO, the cooperative amplification, and low fluorescence background, sensitive and accurate detection of miRNAs has been achieved. The strategy proposed here will offer a new approach for reliable quantification of miRNAs in medical research and early clinical diagnostics. PMID:24823448

  11. Intracavity absorption multiplexed sensor network based on dense wavelength division multiplexing filter.

    PubMed

    Zhang, Haiwei; Lu, Ying; Duan, Liangcheng; Zhao, Zhiqiang; Shi, Wei; Yao, Jianquan

    2014-10-01

    We report the system design and experimental verification of an intracavity absorption multiplexed sensor network with hollow core photonic crystal fiber (HCPCF) sensors and dense wavelength division multiplexing (DWDM) filters. Compared with fiber Bragg grating (FBG), it is easier for the DWDM to accomplish a stable output. We realize the concentration detection of three gas cells filled with acetylene. The sensitivity is up to 100 ppmV at 1536.71 nm. Voltage gradient is firstly used to optimize the intracavity sensor network enhancing the detection efficiency up to 6.5 times. To the best of our knowledge, DWDM is firstly used as a wavelength division multiplexing device to realize intracavity absorption multiplexed sensor network. It make it possible to realize high capacity intracavity sensor network via multiplexed technique. PMID:25322029

  12. Sensitive detection of melanoma metastasis using circulating microRNA expression profiles.

    PubMed

    Shiiyama, Rie; Fukushima, Satoshi; Jinnin, Masatoshi; Yamashita, Junji; Miyashita, Azusa; Nakahara, Satoshi; Kogi, Ai; Aoi, Jun; Masuguchi, Shinichi; Inoue, Yuji; Ihn, Hironobu

    2013-10-01

    Numerous studies have indicated that the serum levels of microRNAs are useful for the diagnosis or evaluation of activity in human diseases. However, determining the level of only one of the nearly 2000 microRNAs identified so far may be less significant. Accordingly, we examined the possibility that the expression pattern of multiple microRNAs in each patient may be a more reliable disease marker for melanoma, especially metastatic disease, focusing on the interaction among microRNAs. Six microRNAs (miR-9, miR-145, miR-150, miR-155, miR-203, and miR-205) were evaluated using real-time PCR in 11 patients with metastatic melanoma and in 16 patients without melanoma. The expression of the six microRNAs was significantly different between the patients with metastasis and those without it. MiR-9 and miR-205 and miR-203 and miR-205 showed significant correlations, and the combination of miR-9, miR-145, miR-150, miR-155, and miR-205 was more sensitive than when each miR was used individually to distinguish the patients with metastasis from those without it. This is the first report demonstrating the expression profiles of multiple microRNAs in melanoma patients. Clarifying the involvement of the microRNA network in the pathogenesis of melanoma may contribute to the development of new diagnostic tools and to advancing the understanding of this disease.

  13. Detection of cyclin D1 mRNA by hybridization sensitive NIC-oligonucleotide probe.

    PubMed

    Kovaliov, Marina; Segal, Meirav; Kafri, Pinhas; Yavin, Eylon; Shav-Tal, Yaron; Fischer, Bilha

    2014-05-01

    A large group of fluorescent hybridization probes, includes intercalating dyes for example thiazole orange (TO). Usually TO is coupled to nucleic acids post-synthetically which severely limits its use. Here, we have developed a phosphoramidite monomer, 10, and prepared a 2'-OMe-RNA probe, labeled with 5-(trans-N-hexen-1-yl-)-TO-2'-deoxy-uridine nucleoside, dU(TO), (Nucleoside bearing an Inter-Calating moiety, NIC), for selective mRNA detection. We investigated a series of 15-mer 2'-OMe-RNA probes, targeting the cyclin D1 mRNA, containing one or several dU(TO) at various positions. dU(TO)-2'-OMe-RNA exhibited up to 7-fold enhancement of TO emission intensity upon hybridization with the complementary RNA versus that of the oligomer alone. This NIC-probe was applied for the specific detection of a very small amount of a breast cancer marker, cyclin D1 mRNA, in total RNA extract from cancerous cells (250 ng/μl). Furthermore, this NIC-probe was found to be superior to our related NIF (Nucleoside with Intrinsic Fluorescence)-probe which could detect cyclin D1 mRNA target only at high concentrations (1840 ng/μl). Additionally, dU(T) can be used as a monomer in solid-phase oligonucleotide synthesis, thus avoiding the need for post-synthetic modification of oligonucleotide probes. Hence, we propose dU(TO) oligonucleotides, as hybridization probes for the detection of specific RNA in homogeneous solutions and for the diagnosis of breast cancer.

  14. Development of a sensitive and specific multiplex PCR method for the simultaneous detection of chicken, duck and goose DNA in meat products.

    PubMed

    Hou, Bo; Meng, Xianrong; Zhang, Liyuan; Guo, Jinyue; Li, Shaowen; Jin, Hui

    2015-03-01

    Identifying the origin of animal species in manufactured meat products is of considerable economic, religious and sanitary importance. In this study, we developed a multiplex PCR method to simultaneously detect chicken, duck and goose DNA in meat products derived from beef, pork, mutton or quail. The PCR primers were designed based on the sequence of mitochondrial genes of each avian species, and the amplicon sizes were 131, 283 and 387bp for chicken, duck and goose, respectively. The method had no cross-reaction with DNA isolated from beef, mutton, pork or quail, and generated products at a target DNA content as low as 0.05ng, or a target meat content of 1% of total meat weight. Moreover, screening of 24 commercial meat samples using this method indicated that six, two and one samples were contaminated with chicken, duck, or both, respectively, suggesting its usefulness for the simultaneous identification of chicken, duck and goose DNA in commercial meat products.

  15. Silencing MRP1-4 genes by RNA interference enhances sensitivity of human hepatoma cells to chemotherapy

    PubMed Central

    Su, Zheng; Liu, Gaojie; Fang, Tingfeng; Wang, Yang; Zhang, Huayao; Yang, Shanglin; Wei, Jinxing; Lv, Zejian; Tan, Langping; Liu, Jianping

    2016-01-01

    Aim: Besides surgical treatment, systematic chemotherapy plays a crucial role in HCC treatment, especially for patients with advanced HCC. However, none of the single-drug-treatment strategies have shown significant survival benefit due to a high incidence rate of chemoresistance. This study was designed to observe the effect of small interfering of RNA (SiRNA) targeting multidrug resistance-related protein 1-4 (MRP1, MRP2, MRP3, and MRP4) in modulating drug resistance of HepG2/ADM and SMMC7721/ADM cells. Methods: HepG2/Adriamycin (ADM) and SMMC7721/ADM cell lines were developed by exposing parental cells to stepwise increasing concentrations of ADM. MTT assay was used to determine drug sensitivity and half inhibitory concentration (IC50) of drugs was calculated. Flow cytometry was employed to analyze cell cycle distribution. MRP1-4 mRNA expression levels were measured by quantitative real-time PCR (QRT-PCR). Expression of proteins was analyzed by Western blot. The growth curve was draw and the cell apoptosis was also observed. Animal experiment was used to compare the cell growth. Results: MTT assay showed that the values of IC50 and RI of HepG2/ADM and SMMC7721/ADM decreased after siRNA treatment in HepG2/ADM cells and SMMC7721/ADM cells. QRT-PCR analysis demonstrated the MRP1-4 mRNA expression decreased significantly in HepG2/ADM cells and SMMC7721/ADM cells after siRNA transfection. In addition, compared with parental cells, MRP1-4 protein expressions apparently decreased in SMMC7721/ADM and HepG2/ADM cells. Flow cytometry showed significantly elevated apoptosis rate following MRP1-4 siRNA transfection. Animal experiment suggested that silencing MRP1-4 gene in vivo inhibited tumor growth. Conclusion: Inhibition of MRP1-4 by small interfering RNA enhanced and selectively restored sensitivity of hepatoma cells to drugs. MRP1-4 siRNA might represent a new therapeutic option for HCC. PMID:27398162

  16. p53-Dependent DNA damage response sensitive to editing-defective tRNA synthetase in zebrafish.

    PubMed

    Song, Youngzee; Shi, Yi; Carland, Tristan M; Lian, Shanshan; Sasaki, Tomoyuki; Schork, Nicholas J; Head, Steven R; Kishi, Shuji; Schimmel, Paul

    2016-07-26

    Brain and heart pathologies are caused by editing defects of transfer RNA (tRNA) synthetases, which preserve genetic code fidelity by removing incorrect amino acids misattached to tRNAs. To extend understanding of the broader impact of synthetase editing reactions on organismal homeostasis, and based on effects in bacteria ostensibly from small amounts of mistranslation of components of the replication apparatus, we investigated the sensitivity to editing of the vertebrate genome. We show here that in zebrafish embryos, transient overexpression of editing-defective valyl-tRNA synthetase (ValRS(ED)) activated DNA break-responsive H2AX and p53-responsive downstream proteins, such as cyclin-dependent kinase (CDK) inhibitor p21, which promotes cell-cycle arrest at DNA damage checkpoints, and Gadd45 and p53R2, with pivotal roles in DNA repair. In contrast, the response of these proteins to expression of ValRS(ED) was abolished in p53-deficient fish. The p53-activated downstream signaling events correlated with suppression of abnormal morphological changes caused by the editing defect and, in adults, reversed a shortened life span (followed for 2 y). Conversely, with normal editing activities, p53-deficient fish have a normal life span and few morphological changes. Whole-fish deep sequencing showed genomic mutations associated with the editing defect. We suggest that the sensitivity of p53 to expression of an editing-defective tRNA synthetase has a critical role in promoting genome integrity and organismal homeostasis.

  17. In Organello Transcription in Maize Mitochondria and Its Sensitivity to Inhibitors of RNA Synthesis 1

    PubMed Central

    Finnegan, Patrick M.; Brown, Gregory G.

    1987-01-01

    Purified mitochondrial preparations from etiolated maize shoots support the incorporation of radioactivity from labeled UTP into RNA. The incorporation is linear with time for up to 2 hours, shows Michaelis-Menton kinetics with respect to the concentration of the labeled substrate, UTP, and has salt and pH optima which are different than those previously reported for RNA synthesis by isolated chloroplasts. When a crude mitochondrial preparation is subjected to isopycnic sucrose gradient centrifugation, the bulk of the RNA synthetic activity co-sediments with mitochondrial marker enzymes and with the mitochondrial 26S and 18S rRNAs. Maize mitochondrial RNA synthesis is prevented by actinomycin D and ethidium bromide but unaffected by α-amanitin. It is strongly inhibited by rifampicin at concentrations which have no effect on nuclear and chloroplast RNA synthesis, but only moderately inhibited by rifampicin at concentrations which completely inhibit bacterial RNA synthesis. The optimization, cell fractionation, and inhibitor data all suggest that contaminating organelles and bacteria do not contribute appreciably to the RNA synthesis in purified mitochondrial preparations. Images Fig. 4 PMID:16665676

  18. MicroRNA-145 Modulates Tumor Sensitivity to Radiation in Prostate Cancer.

    PubMed

    Gong, Pijun; Zhang, Tingting; He, Dalin; Hsieh, Jer-Tsong

    2015-12-01

    Radiation therapy prior to surgery has increasingly become the standard of care for locally advanced prostate cancer, however tumor radioresistance remains a major clinical problem. While restoration of microRNA-145 (miR-145) expression reduces chemoradioresistance in glioblastoma and suppress prostate cancer proliferation, migration and invasion, the role of miR-145 in response to radiation therapy for prostate cancer is still unknown. The aim of this study was to investigate the role of miR-145 in determining the tumor response to radiation treatment in prostate cancer. Human prostate cancer cells LNCAP and PC3 were transfected with miR-145 mimic. Clonogenic assay was used to determine whether overexpression of miR-145 could alter radiation response in vitro. Immunofluorescence of γ-H2AX and flow cytometric analysis of phosphorylated histone H3 were performed to investigate the potential mechanisms contributing to the enhanced radiation-induced cell killing induced by miR-145. In addition, a qPCR-based array was used to detect the possible miR-145-mediated regulated genes involved. Tumor growth delay assays and survival curves were then analyzed in an animal model to investigate whether miR-145 induced radiosensitivity in vivo. Furthermore, miR-145 expression was assessed in 30 prostate tumor tissue biopsies taken prior to neoadjuvant radiotherapy using miRNA arrays. Our current study suggested that ectopic expression of miR-145 significantly sensitized prostate cancer cells to radiation and we used γ-H2AX phosphorylation as a surrogate marker of radiotherapy response versus miR-145 expression levels. We observed significantly more foci per cell in the group treated with miR-145 and radiation. In addition, mitotic catastrophe was significantly increased in cells receiving miR-145 and radiation. The above results suggest that miR-145 appears to reduced the efficiency of the repair of radiation-induced DNA double-strand breaks in cells. A detailed examination of

  19. Multiplex PCR Tests for Detection of Pathogens Associated with Gastroenteritis

    PubMed Central

    Zhang, Hongwei; Morrison, Scott; Tang, Yi-Wei

    2016-01-01

    Synopsis A wide range of enteric pathogens can cause infectious gastroenteritis. Conventional diagnostic algorithms including culture, biochemical identification, immunoassay and microscopic examination are time consuming and often lack sensitivity and specificity. Advances in molecular technology have as allowed its use as clinical diagnostic tools. Multiplex PCR based testing has made its way to gastroenterology diagnostic arena in recent years. In this article we present a review of recent laboratory developed multiplex PCR tests and current commercial multiplex gastrointestinal pathogen tests. We will focus on two FDA cleared commercial syndromic multiplex tests: Luminex xTAG GPP and Biofire FimArray GI test. These multiplex tests can detect and identify multiple enteric pathogens in one test and provide results within hours. Multiplex PCR tests have shown superior sensitivity to conventional methods for detection of most pathogens. The high negative predictive value of these multiplex tests has led to the suggestion that they be used as screening tools especially in outbreaks. Although the clinical utility and benefit of multiplex PCR test are to be further investigated, implementing these multiplex PCR tests in gastroenterology diagnostic algorithm has the potential to improve diagnosis of infectious gastroenteritis. PMID:26004652

  20. Weighted multiplex networks.

    PubMed

    Menichetti, Giulia; Remondini, Daniel; Panzarasa, Pietro; Mondragón, Raúl J; Bianconi, Ginestra

    2014-01-01

    One of the most important challenges in network science is to quantify the information encoded in complex network structures. Disentangling randomness from organizational principles is even more demanding when networks have a multiplex nature. Multiplex networks are multilayer systems of [Formula: see text] nodes that can be linked in multiple interacting and co-evolving layers. In these networks, relevant information might not be captured if the single layers were analyzed separately. Here we demonstrate that such partial analysis of layers fails to capture significant correlations between weights and topology of complex multiplex networks. To this end, we study two weighted multiplex co-authorship and citation networks involving the authors included in the American Physical Society. We show that in these networks weights are strongly correlated with multiplex structure, and provide empirical evidence in favor of the advantage of studying weighted measures of multiplex networks, such as multistrength and the inverse multiparticipation ratio. Finally, we introduce a theoretical framework based on the entropy of multiplex ensembles to quantify the information stored in multiplex networks that would remain undetected if the single layers were analyzed in isolation.

  1. Rapid and sensitive suspension array for multiplex detection of organophosphorus pesticides and carbamate pesticides based on silica-hydrogel hybrid microbeads.

    PubMed

    Wang, Xuan; Mu, Zhongde; Shangguan, Fengqi; Liu, Ran; Pu, Yuepu; Yin, Lihong

    2014-05-30

    A technique for multiplex detection of organophosphorus pesticides and carbamate pesticides has been developed using a suspension array based on silica-hydrogel hybrid microbeads (SHHMs). The main advantage of SHHMs, which consist of both silica and hydrogel materials, is that they not only could be distinguished by their characteristic reflection peak originating from the stop-band of the photonic crystal but also have low non-specific adsorption of proteins. Using fluorescent immunoassay, the LODs for fenitrothion, chlorpyrifos-methyl, fenthion, carbaryl and metolcarb were measured to be 0.02ng/mL, 0.012ng/mL, 0.04ng/mL, 0.05ng/mL and 0.1ng/mL, respectively, all of which are much lower than the maximum residue limits, as reported in the European Union pesticides database. All the determination coefficients for these five pesticides were greater than 0.99, demonstrating excellent correlations. The suspension array was specific and had no significant cross-reactivity with other chemicals. The results for the detection of pesticide residues collected from agricultural samples using this method agree well with those from liquid chromatography-tandem mass spectrometry. Our results showed that this simple method is suitable for simultaneous detection of these five pesticides residues in fruits and vegetables.

  2. Rapid Multiplexed Flow Cytometric Assay for Botulinum Neurotoxin Detection Using an Automated Fluidic Microbead-Trapping Flow Cell for Enhanced Sensitivity

    SciTech Connect

    Ozanich, Richard M.; Bruckner-Lea, Cindy J.; Warner, Marvin G.; Miller, Keith D.; Antolick, Kathryn C.; Marks, James D.; Lou, Jianlong; Grate, Jay W.

    2009-07-15

    A bead-based sandwich immunoassay for botulinum neurotoxin serotype A (BoNT/A) has been developed and demonstrated using a recombinant 50 kDa fragment (BoNT/A-HC-fragment) of the BoNT/A heavy chain (BoNT/A-HC) as a structurally valid simulant. Three different anti-BoNT/A antibodies were attached to three different fluorescent dye encoded flow cytometry beads for multiplexing. The assay was conducted in two formats: a manual microcentrifuge tube format and an automated fluidic system format. Flow cytometry detection was used for both formats. The fluidic system used a novel microbead-trapping flow cell to capture antibody-coupled beads with subsequent sequential perfusion of sample, wash, dye-labeled reporter antibody, and final wash solutions. After the reaction period, the beads were collected for analysis by flow cytometry. Sandwich assays performed on the fluidic system gave median fluorescence intensity signals on the flow cytometer that were 2-4 times higher than assays performed manually in the same amount of time. Limits of detection were estimated at 1 pM (~50 pg/mL for BoNT/A-HC-fragment) for the 15 minute fluidic assay.

  3. Nodamura virus B2 amino terminal domain sensitivity to small interfering RNA.

    PubMed

    Ali, P Shaik Syed; John, Jasmine; Selvaraj, Manikandan; Kek, Teh Lay; Salleh, Mohd Zaki

    2015-05-01

    Nodamura virus (NoV) B2, a suppressor of RNA interference, binds double stranded RNAs (dsRNAs) and small interfering RNAs (siRNAs) corresponding to Dicer substrates and products. Here, we report that the amino terminal domain of NoV B2 (NoV B2 79) specifically binds siRNAs but not dsRNAs. NoV B2 79 oligomerizes on binding to 27 nucleotide siRNA. Mutation of the residues phenylalanine49 and alanine60 to cysteine and methionine, respectively enhances the RNA binding affinity of NoV B2 79. Circular dichroism spectra demonstrated that the wild type and mutant NoV B2 79 have similar secondary structure conformations.

  4. Insulin regulates enzyme activity, malonyl-CoA sensitivity and mRNA abundance of hepatic carnitine palmitoyltransferase-I.

    PubMed Central

    Park, E A; Mynatt, R L; Cook, G A; Kashfi, K

    1995-01-01

    The regulation of hepatic mitochondrial carnitine palmitoyltransferase-I (CPT-I) was studied in rats during starvation and insulin-dependent diabetes and in rat H4IIE cells. The Vmax. for CPT-I in hepatic mitochondrial outer membranes isolated from starved and diabetic rats increased 2- and 3-fold respectively over fed control values with no change in Km values for substrates. Regulation of malonyl-CoA sensitivity of CPT-I in isolated mitochondrial outer membranes was indicated by an 8-fold increase in Ki during starvation and by a 50-fold increase in Ki in the diabetic state. Peroxisomal and microsomal CPT also had decreased sensitivity to inhibition by malonyl-CoA during starvation. CPT-I mRNA abundance was 7.5 times greater in livers of 48-h-starved rats and 14.6 times greater in livers of insulin-dependent diabetic rats compared with livers of fed rats. In H4IIE cells, insulin increased CPT-I sensitivity to inhibition by malonyl-CoA in 4 h, and sensitivity continued to increase up to 24 h after insulin addition. CPT-I mRNA levels in H4IIE cells were decreased by insulin after 4 h and continued to decrease so that at 24 h there was a 10-fold difference. The half-life of CPT-I mRNA was 4 h in the presence of actinomycin D or with actinomycin D plus insulin. These results suggest that insulin regulates CPT-I by inhibiting transcription of the CPT-I gene. Images Figure 2 Figure 4 PMID:7575418

  5. miRNA-195 sensitizes human hepatocellular carcinoma cells to 5-FU by targeting BCL-w.

    PubMed

    Yang, Xiaoyan; Yin, Jie; Yu, Jia; Xiang, Qiong; Liu, Yunmei; Tang, Shensong; Liao, Duanfang; Zhu, Bingyang; Zu, Xuyu; Tang, Huifang; Lei, Xiaoyong

    2012-01-01

    The role of microRNA-195 in developing acquired drug resistance in hepatocellular carcinoma cells was investigated. Expression profiling of miRNAs revealed a limited set of miRNAs with altered expression in drug resistant hepatocellular carcinoma cell line BEL-7402/5-FU compared to its parental BEL-7402 cell line. Real-time PCR confirmed down-regulation of miRNA-195 in BEL-7402/5-FU cells. Overexpression of miRNA-195 sensitized BEL-7402/5-FU cells to anticancer drugs. Consistent with these findings, miR-195 antisense oligonucleotide induced drug resistance in BEL-7402/5-FU cells. Also, the basal levels of the anti-apoptotic protein Bcl-w were high in BEL-7402/5-FU cells and miR-195 overexpression repressed Bcl-w protein level and inhibited the luciferase activity of a Bcl-w 3' untranslated region-based reporter construct in both BEL-7402/5-FU and BEL-7402 cells. These results indicate that miR-195 could improve the drug sensitivity at least in part by targeting Bcl-w to increase cell apoptosis in hepatocellular carcinoma cells.

  6. An electrochemical biosensor for sensitive detection of microRNA-155: combining target recycling with cascade catalysis for signal amplification.

    PubMed

    Wu, Xiaoyan; Chai, Yaqin; Zhang, Pu; Yuan, Ruo

    2015-01-14

    In this work, a new electrochemical biosensor based on catalyzed hairpin assembly target recycling and cascade electrocatalysis (cytochrome c (Cyt c) and alcohol oxidase (AOx)) for signal amplification was constructed for highly sensitive detection of microRNA (miRNA). It is worth pointing out that target recycling was achieved only based on strand displacement process without the help of nuclease. Moreover, porous TiO2 nanosphere was synthesized, which could offer more surface area for Pt nanoparticles (PtNPs) enwrapping and enhance the amount of immobilized DNA strand 1 (S1) and Cyt c accordingly. With the mimicking sandwich-type reaction, the cascade catalysis amplification strategy was carried out by AOx catalyzing ethanol to acetaldehyde with the concomitant formation of high concentration of H2O2, which was further electrocatalyzed by PtNPs and Cyt c. This newly designed biosensor provided a sensitive detection of miRNA-155 from 0.8 fM to 1 nM with a relatively low detection limit of 0.35 fM.

  7. pH-Sensitive siRNA nanovector for targeted gene silencing and cytotoxic effect in cancer cells.

    PubMed

    Mok, Hyejung; Veiseh, Omid; Fang, Chen; Kievit, Forrest M; Wang, Freddy Y; Park, James O; Zhang, Miqin

    2010-12-01

    A small interfering RNA (siRNA) nanovector with dual targeting specificity and dual therapeutic effect is developed for targeted cancer imaging and therapy. The nanovector is composed of an iron oxide magnetic nanoparticle core coated with three different functional molecules: polyethyleneimine (PEI), siRNA, and chlorotoxin (CTX). The primary amine group of PEI is blocked with citraconic anhydride that is removable at acidic conditions, not only to increase its biocompatibility at physiological conditions but also to elicit a pH-sensitive cytotoxic effect in the acidic tumor microenvironment. The PEI is covalently immobilized on the nanovector via a disulfide linkage that is cleavable after cellular internalization of the nanovector. CTX as a tumor-specific targeting ligand and siRNA as a therapeutic payload are conjugated on the nanovector via a flexible and hydrophilic PEG linker for targeted gene silencing in cancer cells. With a size of ∼60 nm, the nanovector exhibits long-term stability and good magnetic property for magnetic resonance imaging. The multifunctional nanovector exhibits both significant cytotoxic and gene silencing effects at acidic pH conditions for C6 glioma cells, but not at physiological pH conditions. Our results suggest that this nanovector system could be safely used as a potential therapeutic agent for targeted treatment of glioma as well as other cancers.

  8. [Application of Cationic Aluminum Phthalocyanine, a Red-Emitting Fluorescent Probe, for Sensitive Quantitative Analysis of RNA at Nanogram Level].

    PubMed

    Guo, Meng-lin; Yang, Hui-qing; Huang, Ping; Chen, Lin; Li, Dong-hui

    2016-03-01

    Tetrasubstituted trimethyl ammonium iodide aluminum phthalocyanine (TTMAAlPc), a positively charged phthalocyanine compound, is an emerging and potentially useful red-emitting fluorescence probe. The study showed that the fluorescence of TTMAAlPc could be quenched by RNA with high efficiency in weak alkaline media, and the degree of quenching has a linear relationship with RNA in a wide concentration range. The mechanism of quenching behavior of RNA on TTMAAlPc was discussed. It was attributed by the static interaction between RNA and TTMAAlPc, and the assembly of TTMAAlPc induced by RNA. Based on this new discovery, a novel method for quantitative determination of RNA at nanogram level has been established. The factors, including the pH of medium, buffer system, reaction time, reaction temperature, the usage of TTMAAlPc as well as the interferences, which affected the determination, were investigated and discussed. Under optimum conditions, the linear range of the calibration curve was 7.71-1 705.57 ng x mL(-1). The detection limit for RNA was 1.55 ng x mL(-1). This method has been applied to the analysis of practical samples with satisfied results. The constructed method is of high sensitivity and has a wide linear range, it also showed strong ability in the tolerance of foreign substances from anions, cations, surfactants and vitamins, all of which are common interferences encountered in the determination of RNA. Besides, it is the first report that the fluorescence quantum yield of TTMAAlPc has been measured at different pH by reference method in this work. The achieved data indicated that the fluorescence quantum yield of TTMAAlPc is larger than 20% and it keeps constant in a wide range of acidity, implying that TTMAAlPc is a high-quality red-emitting fluorescence probe, it has great potential for practical applications, thus is worthy of further study. This work expands the application of phthalocyanine compound in analytical sciences.

  9. [Application of Cationic Aluminum Phthalocyanine, a Red-Emitting Fluorescent Probe, for Sensitive Quantitative Analysis of RNA at Nanogram Level].

    PubMed

    Guo, Meng-lin; Yang, Hui-qing; Huang, Ping; Chen, Lin; Li, Dong-hui

    2016-03-01

    Tetrasubstituted trimethyl ammonium iodide aluminum phthalocyanine (TTMAAlPc), a positively charged phthalocyanine compound, is an emerging and potentially useful red-emitting fluorescence probe. The study showed that the fluorescence of TTMAAlPc could be quenched by RNA with high efficiency in weak alkaline media, and the degree of quenching has a linear relationship with RNA in a wide concentration range. The mechanism of quenching behavior of RNA on TTMAAlPc was discussed. It was attributed by the static interaction between RNA and TTMAAlPc, and the assembly of TTMAAlPc induced by RNA. Based on this new discovery, a novel method for quantitative determination of RNA at nanogram level has been established. The factors, including the pH of medium, buffer system, reaction time, reaction temperature, the usage of TTMAAlPc as well as the interferences, which affected the determination, were investigated and discussed. Under optimum conditions, the linear range of the calibration curve was 7.71-1 705.57 ng x mL(-1). The detection limit for RNA was 1.55 ng x mL(-1). This method has been applied to the analysis of practical samples with satisfied results. The constructed method is of high sensitivity and has a wide linear range, it also showed strong ability in the tolerance of foreign substances from anions, cations, surfactants and vitamins, all of which are common interferences encountered in the determination of RNA. Besides, it is the first report that the fluorescence quantum yield of TTMAAlPc has been measured at different pH by reference method in this work. The achieved data indicated that the fluorescence quantum yield of TTMAAlPc is larger than 20% and it keeps constant in a wide range of acidity, implying that TTMAAlPc is a high-quality red-emitting fluorescence probe, it has great potential for practical applications, thus is worthy of further study. This work expands the application of phthalocyanine compound in analytical sciences. PMID:27400518

  10. High Sensitivity RT-qPCR Assay of Nonlabeled siRNA in Small Blood Volume for Pharmacokinetic Studies: Application to Survivin siRNA.

    PubMed

    Yeung, Bertrand Z; Lu, Ze; Wientjes, Guillaume M; Au, Jessie L-S

    2015-11-01

    RNAi therapeutics provide an opportunity to correct faulty genes, and several RNAi have entered clinical evaluation. The existing quantification methods typically use radioactivity- or fluorescence-labeled RNAi, require large blood volumes, and/or have a limited dynamic detection range. We established a quantitative reverse transcriptase real-time polymerase chain reaction (RT-qPCR) assay to measure RNAi; the model analyte was survivin siRNA (siSurvivin). A second siRNA was used as the internal standard. The three major steps were (a) extraction of the two siRNAs from blood or water, (b) synthesis of their cDNA by poly-A extension, and (c) qPCR of cDNA. Standard curves were established. Utility of the assay was demonstrated in a pharmacokinetic study where all 12 samples for the blood concentration-time profile were obtained from a single mouse given an intravenous dose of 1 nmole siSurvivin (prepared as lipoplex with pegylated cationic liposomes). The RT-qPCR assay was sensitive (lower detection limit of 100 fM) and had a 5 × 107-fold dynamic range and low sample volume requirement (10 μL). The 16-point standard curves constructed using whole blood samples were linear (R (2) > 0.98). The intraday and interday variations for the slopes were ≤6%, although the variations for accuracy and precision at individual concentrations were substantially higher (58-145%). Standard curves prepared with water in place of blood showed similar results (<6% difference), indicating water may be used when blood is not available. The current RT-qPCR assay enabled the measurement of nonlabeled siRNA in small volume of blood samples. PMID:26286676

  11. The Temperature Sensitivity of a Mutation in the Essential tRNA Modification Enzyme tRNA Methyltransferase D (TrmD)*

    PubMed Central

    Masuda, Isao; Sakaguchi, Reiko; Liu, Cuiping; Gamper, Howard; Hou, Ya-Ming

    2013-01-01

    Conditional temperature-sensitive (ts) mutations are important reagents to study essential genes. Although it is commonly assumed that the ts phenotype of a specific mutation arises from thermal denaturation of the mutant enzyme, the possibility also exists that the mutation decreases the enzyme activity to a certain level at the permissive temperature and aggravates the negative effect further upon temperature upshifts. Resolving these possibilities is important for exploiting the ts mutation for studying the essential gene. The trmD gene is essential for growth in bacteria, encoding the enzyme for converting G37 to m1G37 on the 3′ side of the tRNA anticodon. This conversion involves methyl transfer from S-adenosyl methionine and is critical to minimize tRNA frameshift errors on the ribosome. Using the ts-S88L mutation of Escherichia coli trmD as an example, we show that although the mutation confers thermal lability to the enzyme, the effect is relatively minor. In contrast, the mutation decreases the catalytic efficiency of the enzyme to 1% at the permissive temperature, and at the nonpermissive temperature, it renders further deterioration of activity to 0.1%. These changes are accompanied by losses of both the quantity and quality of tRNA methylation, leading to the potential of cellular pleiotropic effects. This work illustrates the principle that the ts phenotype of an essential gene mutation can be closely linked to the catalytic defect of the gene product and that such a mutation can provide a useful tool to study the mechanism of catalytic inactivation. PMID:23986443

  12. p53-Dependent DNA damage response sensitive to editing-defective tRNA synthetase in zebrafish.

    PubMed

    Song, Youngzee; Shi, Yi; Carland, Tristan M; Lian, Shanshan; Sasaki, Tomoyuki; Schork, Nicholas J; Head, Steven R; Kishi, Shuji; Schimmel, Paul

    2016-07-26

    Brain and heart pathologies are caused by editing defects of transfer RNA (tRNA) synthetases, which preserve genetic code fidelity by removing incorrect amino acids misattached to tRNAs. To extend understanding of the broader impact of synthetase editing reactions on organismal homeostasis, and based on effects in bacteria ostensibly from small amounts of mistranslation of components of the replication apparatus, we investigated the sensitivity to editing of the vertebrate genome. We show here that in zebrafish embryos, transient overexpression of editing-defective valyl-tRNA synthetase (ValRS(ED)) activated DNA break-responsive H2AX and p53-responsive downstream proteins, such as cyclin-dependent kinase (CDK) inhibitor p21, which promotes cell-cycle arrest at DNA damage checkpoints, and Gadd45 and p53R2, with pivotal roles in DNA repair. In contrast, the response of these proteins to expression of ValRS(ED) was abolished in p53-deficient fish. The p53-activated downstream signaling events correlated with suppression of abnormal morphological changes caused by the editing defect and, in adults, reversed a shortened life span (followed for 2 y). Conversely, with normal editing activities, p53-deficient fish have a normal life span and few morphological changes. Whole-fish deep sequencing showed genomic mutations associated with the editing defect. We suggest that the sensitivity of p53 to expression of an editing-defective tRNA synthetase has a critical role in promoting genome integrity and organismal homeostasis. PMID:27402763

  13. microRNA160 dictates stage-specific auxin and cytokinin sensitivities and directs soybean nodule development.

    PubMed

    Nizampatnam, Narasimha Rao; Schreier, Spencer John; Damodaran, Suresh; Adhikari, Sajag; Subramanian, Senthil

    2015-10-01

    Legume nodules result from coordinated interactions between the plant and nitrogen-fixing rhizobia. The phytohormone cytokinin promotes nodule formation, and recent findings suggest that the phytohormone auxin inhibits nodule formation. Here we show that microRNA160 (miR160) is a key signaling element that determines the auxin/cytokinin balance during nodule development in soybean (Glycine max). miR160 appears to promote auxin activity by suppressing the levels of the ARF10/16/17 family of repressor ARF transcription factors. Using quantitative PCR assays and a fluorescence miRNA sensor, we show that miR160 levels are relatively low early during nodule formation and high in mature nodules. We had previously shown that ectopic expression of miR160 in soybean roots led to a severe reduction in nodule formation, coupled with enhanced sensitivity to auxin and reduced sensitivity to cytokinin. Here we show that exogenous cytokinin restores nodule formation in miR160 over-expressing roots. Therefore, low miR160 levels early during nodule development favor cytokinin activity required for nodule formation. Suppression of miR160 levels using a short tandem target mimic (STTM160) resulted in reduced sensitivity to auxin and enhanced sensitivity to cytokinin. In contrast to miR160 over-expressing roots, STTM160 roots had increased nodule formation, but nodule maturation was significantly delayed. Exogenous auxin partially restored proper nodule formation and maturation in STTM160 roots, suggesting that high miR160 activity later during nodule development favors auxin activity and promotes nodule maturation. Therefore, miR160 dictates developmental stage-specific sensitivities to auxin and cytokinin to direct proper nodule formation and maturation in soybean.

  14. Multiplex polymerase chain reaction tests for detection of pathogens associated with gastroenteritis.

    PubMed

    Zhang, Hongwei; Morrison, Scott; Tang, Yi-Wei

    2015-06-01

    A wide range of enteric pathogens can cause infectious gastroenteritis. Conventional diagnostic algorithms are time-consuming and often lack sensitivity and specificity. Advances in molecular technology have provided new clinical diagnostic tools. Multiplex polymerase chain reaction (PCR)-based testing has been used in gastroenterology diagnostics in recent years. This article presents a review of recent laboratory-developed multiplex PCR tests and current commercial multiplex gastrointestinal pathogen tests. It focuses on two commercial syndromic multiplex tests: Luminex xTAG Gastrointestinal Pathogen Panel and BioFire FilmArray gastrointestinal test. Multiplex PCR tests have shown superior sensitivity to conventional methods for detection of most pathogens.

  15. Stranded Whole Transcriptome RNA-Seq for All RNA Types

    PubMed Central

    Yan, Pearlly X.; Fang, Fang; Buechlein, Aaron; Ford, James B.; Tang, Haixu; Huang, Tim H.; Burow, Matthew E.; Liu, Yunlong; Rusch, Douglas B.

    2015-01-01

    Stranded whole transcriptome RNA-Seq described in this unit captures quantitative expression data for all types of RNA including, but not limited to miRNA (microRNA), piRNA (Piwi-interacting RNA), snoRNA (small nucleolar RNA), lincRNA (large non-coding intergenic RNA), SRP RNA (signal recognition particle RNA), tRNA (transfer RNA), mtRNA (mitochondrial RNA) and mRNA (messenger RNA). The size and nature of these types of RNA are irrelevant to the approach described here. Barcoded libraries for multiplexing on the Illumina platform are generated with this approach but it can be applied to other platforms with a few modifications. PMID:25599667

  16. Stranded Whole Transcriptome RNA-Seq for All RNA Types.

    PubMed

    Miller, David F B; Yan, Pearlly X; Fang, Fang; Buechlein, Aaron; Ford, James B; Tang, Haixu; Huang, Tim H; Burow, Matthew E; Liu, Yunlong; Rusch, Douglas B; Nephew, Kenneth P

    2015-01-20

    Stranded whole transcriptome RNA-Seq described in this unit captures quantitative expression data for all types of RNA including, but not limited to, miRNA (microRNA), piRNA (Piwi-interacting RNA), snoRNA (small nucleolar RNA), lincRNA (large non-coding intergenic RNA), SRP RNA (signal recognition particle RNA), tRNA (transfer RNA), mtRNA (mitochondrial RNA), and mRNA (messenger RNA). The size and nature of these types of RNA are irrelevant to the approach described here. Barcoded libraries for multiplexing on the Illumina platform are generated with this approach but it can be applied to other platforms with a few modifications.

  17. Ultrasound-sensitive siRNA-loaded nanobubbles formed by hetero-assembly of polymeric micelles and liposomes and their therapeutic effect in gliomas.

    PubMed

    Yin, Tinghui; Wang, Ping; Li, Jingguo; Zheng, Rongqin; Zheng, Bowen; Cheng, Du; Li, Ruitang; Lai, Jieyi; Shuai, Xintao

    2013-06-01

    Ultrasound (US)-sensitive nanobubble (NB) which may utilize the physical power of US exposure to improve delivery efficiency to target cells is emerging as one of the most promising nanocarriers for drug delivery. On the basis of successfully fabricating NBs with the ability of passively accumulating in tumor tissue, in this study we synthesized a US-sensitive NB bearing siRNA (siRNA-NB) for tumor therapy via a hetero-assembling strategy using the siRNA-complexed polymeric micelles and gas-cored liposomes. The US exposure-aided siRNA transfection effectively enhanced the gene silencing effect of siRNA-NBs both in vitro and in vivo, which resulted in much elevated level of cancer cell apoptosis. Consequently, significantly improved therapeutic effect was achieved in a nude mouse glioma model, using siRNA-NBs bearing siRNA to target the anti-apoptosis gene sirtuin 2 (SIRT2). These results show that, with the aid of US exposure, the US-sensitive siRNA-NB may be an ideal delivery vector to mediate highly effective RNA interference for tumor treatment.

  18. Development of Real-Time Multiplex Nucleic Acid Sequence-Based Amplification for Detection of Mycoplasma pneumoniae, Chlamydophila pneumoniae, and Legionella spp. in Respiratory Specimens▿

    PubMed Central

    Loens, K.; Beck, T.; Ursi, D.; Overdijk, M.; Sillekens, P.; Goossens, H.; Ieven, M.

    2008-01-01

    Real-time multiplex isothermal nucleic acid sequence-based amplification (NASBA) was developed to detect Mycoplasma pneumoniae, Chlamydophila pneumoniae, and Legionella spp. in respiratory specimens using the NucliSens Basic Kit (bioMérieux, Boxtel, The Netherlands). Oligonucleotide primers were derived from the M. pneumoniae, C. pneumoniae, and Legionella pneumophila 16S rRNA. For real-time detection, molecular beacons were used. Specificity was established on a panel of bacterial strains. The analytical sensitivity of the assay was determined by testing dilutions of wild-type in vitro-generated RNA in water and dilutions of reference strains in lysis buffer or added to pools of respiratory specimens. Subsequently, a limited number of M. pneumoniae-, C. pneumoniae-, and L. pneumophila-positive and -negative clinical specimens were analyzed. Specific detection of the 16S rRNA of the three organisms was achieved. The analytical sensitivity of the multiplex NASBA on spiked respiratory specimens was slightly diminished compared to the results obtained with the single-target (mono) real-time assays. We conclude that the proposed real-time multiplex NASBA assay, although less sensitive than the real-time mono NASBA assay, is a promising tool for the detection of M. pneumoniae, C. pneumoniae, and Legionella spp. in respiratory specimens, regarding handling, speed, and number of samples that can be analyzed in a single run. PMID:18032625

  19. MicroRNA-320a sensitizes tamoxifen-resistant breast cancer cells to tamoxifen by targeting ARPP-19 and ERRγ.

    PubMed

    Lü, Mingrong; Ding, Keshuo; Zhang, Guofeng; Yin, Mianmian; Yao, Guidong; Tian, Hui; Lian, Jie; Liu, Lin; Liang, Meng; Zhu, Tao; Sun, Fei

    2015-01-01

    Tamoxifen represents a major adjuvant therapy to those patients with estrogen receptor-alpha positive breast cancer. However, tamoxifen resistance occurs quite often, either de novo or acquired during treatment. To investigate the role of miR-320a in the development of resistance to tamoxifen, we established tamoxifen-resistant (TamR) models by continually exposing MCF-7 or T47D breast cancer cells to tamoxifen, and identified microRNA(miRNA)-320a as a down-regulated miRNA in tamoxifen resistant cells. Re-expression of miR-320a was sufficient to sensitize TamR cells to tamoxifen by targeting cAMP-regulated phosphoprotein (ARPP-19) and estrogen-related receptor gamma (ERRγ) as well as their downstream effectors, c-Myc and Cyclin D1. Furthermore, progesterone (P4) promoted the expression of miR-320a by repressing c-Myc expression, while estrogen (E2) exerted the opposite effect. These results suggest the potential therapeutic approach for tamoxifen-resistant breast cancer by restorating miR-320a expression or depleting ARPP-19/ERRγ expression. PMID:25736597

  20. Discriminative identification of miRNA let-7 family members with high specificity and sensitivity using rolling circle amplification.

    PubMed

    Zhao, Bin; Song, Jirui; Guan, Yifu

    2015-02-01

    Rolling circle amplification (RCA) is a new method based on virus DNA reproduction, which has been widely used in the field of miRNA detection. However, discrimination of highly homologous miRNAs is a bottleneck in the research of miRNA. In this study, the RCA process was creatively used to conduct the discrimination of miRNAs. Results showed that T4 RNA ligase 2 could reach the highest circularization efficiency during the RCA process with higher specificity. By using RCA technology, a member of highly homologous miRNAs, let-7, could be discriminated at the amount of 2.5 fmol. This sensitivity could not be achieved by using traditional reverse transcription quantitative polymerase chain reaction (RT-qPCR) method. In addition, detection of miRNAs by using RCA could reach the amount limit of fmol with a good linearity. Optimal RCA technology used in this study is better than RT-qPCR in discriminating highly homologous family miRNAs. Results from this study can promote the applications of RCA in clinical diagnosis, environment protection, health care, disease inspection and prevention, and national security.

  1. Luciferase mRNA Transfection of Antigen Presenting Cells Permits Sensitive Nonradioactive Measurement of Cellular and Humoral Cytotoxicity.

    PubMed

    Omokoko, Tana A; Luxemburger, Uli; Bardissi, Shaheer; Simon, Petra; Utsch, Magdalena; Breitkreuz, Andrea; Türeci, Özlem; Sahin, Ugur

    2016-01-01

    Immunotherapy is rapidly evolving as an effective treatment option for many cancers. With the emerging fields of cancer vaccines and adoptive cell transfer therapies, there is an increasing demand for high-throughput in vitro cytotoxicity assays that efficiently analyze immune effector functions. The gold standard (51)Cr-release assay is very accurate but has the major disadvantage of being radioactive. We reveal the development of a versatile and nonradioactive firefly luciferase in vitro transcribed (IVT) RNA-based assay. Demonstrating high efficiency, consistency, and excellent target cell viability, our optimized luciferase IVT RNA is used to transfect dividing and nondividing primary antigen presenting cells. Together with the long-lasting expression and minimal background, the direct measurement of intracellular luciferase activity of living cells allows for the monitoring of killing kinetics and displays paramount sensitivity. The ability to cotransfect the IVT RNA of the luciferase reporter and the antigen of interest into the antigen presenting cells and its simple read-out procedure render the assay high-throughput in nature. Results generated were comparable to the (51)Cr release and further confirmed the assay's ability to measure antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity. The assay's combined simplicity, practicality, and efficiency tailor it for the analysis of antigen-specific cellular and humoral effector functions during the development of novel immunotherapies. PMID:27057556

  2. Luciferase mRNA Transfection of Antigen Presenting Cells Permits Sensitive Nonradioactive Measurement of Cellular and Humoral Cytotoxicity

    PubMed Central

    Omokoko, Tana A.; Luxemburger, Uli; Bardissi, Shaheer; Simon, Petra; Utsch, Magdalena; Breitkreuz, Andrea; Türeci, Özlem; Sahin, Ugur

    2016-01-01

    Immunotherapy is rapidly evolving as an effective treatment option for many cancers. With the emerging fields of cancer vaccines and adoptive cell transfer therapies, there is an increasing demand for high-throughput in vitro cytotoxicity assays that efficiently analyze immune effector functions. The gold standard 51Cr-release assay is very accurate but has the major disadvantage of being radioactive. We reveal the development of a versatile and nonradioactive firefly luciferase in vitro transcribed (IVT) RNA-based assay. Demonstrating high efficiency, consistency, and excellent target cell viability, our optimized luciferase IVT RNA is used to transfect dividing and nondividing primary antigen presenting cells. Together with the long-lasting expression and minimal background, the direct measurement of intracellular luciferase activity of living cells allows for the monitoring of killing kinetics and displays paramount sensitivity. The ability to cotransfect the IVT RNA of the luciferase reporter and the antigen of interest into the antigen presenting cells and its simple read-out procedure render the assay high-throughput in nature. Results generated were comparable to the 51Cr release and further confirmed the assay's ability to measure antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity. The assay's combined simplicity, practicality, and efficiency tailor it for the analysis of antigen-specific cellular and humoral effector functions during the development of novel immunotherapies. PMID:27057556

  3. Dual fluorophore PNA FIT-probes--extremely responsive and bright hybridization probes for the sensitive detection of DNA and RNA.

    PubMed

    Socher, Elke; Knoll, Andrea; Seitz, Oliver

    2012-09-28

    Fluorescently labeled oligonucleotides are commonly employed as probes to detect specific DNA or RNA sequences in homogeneous solution. Useful probes should experience strong increases in fluorescent emission upon hybridization with the target. We developed dual labeled peptide nucleic acid probes, which signal the presence of complementary DNA or RNA by up to 450-fold enhancements of fluorescence intensity. This enabled the very sensitive detection of a DNA target (40 pM LOD), which was detectable at less than 0.1% of the beacon concentration. In contrast to existing DNA-based molecular beacons, this PNA-based method does not require a stem sequence to enforce dye-dye communication. Rather, the method relies on the energy transfer between a "smart" thiazole orange (TO) nucleotide, which requires formation of the probe-target complex in order to become fluorescent, and terminally appended acceptor dyes. To improve upon fluorescence responsiveness the energy pathways were dissected. Hydrophobic, spectrally mismatched dye combinations allowed significant (99.97%) decreases of background emission in the absence of a target. By contrast, spectral overlap between TO donor emission and acceptor excitation enabled extremely bright FRET signals. This and the large apparent Stokes shift (82 nm) suggests potential applications in the detection of specific RNA targets in biogenic matrices without the need of sample pre-processing prior to detection.

  4. MicroRNA-205 increases the sensitivity of docetaxel in breast cancer

    PubMed Central

    CAI, YANG; YAN, XIANG; ZHANG, GUOQING; ZHAO, WEIHONG; JIAO, SHUNCHANG

    2016-01-01

    Chemotherapy has been widely used in breast cancer therapy, but the efficacy of chemotherapy is intimately associated with the sensitivity of therapeutic drugs to breast cancer. Docetaxel is a first-line chemotherapeutic drug in breast cancer treatment, but further improvement to its efficacy has thus far proved difficult. microRNAs (miRs) are a class of endogenous, small, non-coding RNAs, which regulate gene expression at the post-transcriptional level. miR-205, a regulator of HER-3, is reported to be a tumor suppressor in breast cancer. In the present study, the reintroduction of miR-205 is shown to inhibit cell proliferation and clonogenic potential, and increase the sensitivity of MCF-7 and MDA-MB-231 cells to docetaxel. miR-205 also shows a synergistic effect with docetaxel in vivo. The present study provides a novel strategy to increase the sensitivity to docetaxel in breast cancer patients. PMID:26893700

  5. Multicopy suppressors of temperature-sensitive mutations of yeast mRNA capping enzyme.

    PubMed

    Schwer, B; Shuman, S

    1996-01-01

    We have isolated three Saccharomyces cerevisiae genes-CES1, CES2, and CES3-- that, when present in high copy, suppress the ts growth defect caused by mutations in the CEG1 gene encoding mRNA guanylyltransferase (capping enzyme). Molecular characterization of the capping enzyme suppressor genes reveals the following. CES2 is identical to ESP1, a gene required for proper nuclear division. We show by deletion analysis that the 1573-amino acid ESP1 polypeptide is composed of distinct functional domains. The C-terminal portion of ESP1 is essential for cell growth, but dispensable for CES2 activity. The N-terminal half of ESP1, which is sufficient for CES2 function, displays local sequence similarity to the small subunit of the vaccinia virus RNA capping enzyme. This suggests a basis for suppression by physical or functional interaction between the CES2 domain of ESP1 and the yeast guanylyltransferase. CES1 encodes a novel hydrophilic 915-amino acid protein. The amino acid sequence of CES1 is uninformative, except for its extensive similarity to another yeast gene product of unknown function. The CES1 homologue (designated CES4) is also a multicopy suppressor of capping enzyme ts mutations. Neither CES1 nor CES4 is essential for cell growth, and a double deletion mutant is viable. CES3 corresponds to BUD5, which encodes a putative guanine nucleotide exchange factor. We hypothesize that CES1, CES4, and BUD5 may impact on RNA transactions downstream of cap synthesis that are cap dependent in vivo. PMID:8836740

  6. Multiplexed Engineering in Biology.

    PubMed

    Rogers, Jameson K; Church, George M

    2016-03-01

    Biotechnology is the manufacturing technology of the future. However, engineering biology is complex, and many possible genetic designs must be evaluated to find cells that produce high levels of a desired drug or chemical. Recent advances have enabled the design and construction of billions of genetic variants per day, but evaluation capacity remains limited to thousands of variants per day. Here we evaluate biological engineering through the lens of the design–build–test cycle framework and highlight the role that multiplexing has had in transforming the design and build steps. We describe a multiplexed solution to the ‘test’ step that is enabled by new research. Achieving a multiplexed test step will permit a fully multiplexed engineering cycle and boost the throughput of biobased product development by up to a millionfold.

  7. Multiplex gas chromatography

    NASA Technical Reports Server (NTRS)

    Valentin, Jose R.

    1990-01-01

    The principles of the multiplex gas chromatography (GC) technique, which is a possible candidate for chemical analysis of planetary atmospheres, are discussed. Particular attention is given to the chemical modulators developed by present investigators for multiplex GC, namely, the thermal-desorption, thermal-decomposition, and catalytic modulators, as well as to mechanical modulators. The basic technique of multiplex GC using chemical modulators and a mechanical modulator is demonstrated. It is shown that, with the chemical modulators, only one gas stream consisting of the carrier in combination with the components is being analyzed, resulting in a simplified instrument that requires relatively few consumables. The mechanical modulator demonstrated a direct application of multiplex GC for the analysis of gases in atmosphere of Titan at very low pressures.

  8. Multiplex television transmission system

    NASA Technical Reports Server (NTRS)

    Reed, W. R.

    1967-01-01

    Time-multiplexing system enables several cameras to share a single commercial television transmission channel. This system is useful in industries for visually monitoring several operating areas or instrument panels from a remote location.

  9. Enhancing the analytical performance of electrochemical RNA aptamer-based sensors for sensitive detection of aminoglycoside antibiotics.

    PubMed

    Schoukroun-Barnes, Lauren R; Wagan, Samiullah; Wagan, Samuillah; White, Ryan J

    2014-01-21

    Folding-based electrochemical sensors utilizing structure-switching aptamers are specific, selective, sensitive, and widely applicable to the detection of a variety of target analytes. The specificity is achieved by the binding properties of an electrode-bound RNA or DNA aptamer biorecognition element. Signaling in this class of sensors arises from changes in electron transfer efficiency upon target-induced changes in the conformation/flexibility of the aptamer probe. These changes can be readily monitored electrochemically. Because of this signaling mechanism, there are several approaches to maximizing the analytical attributes (i.e., sensitivity, limit of detection, and observed binding affinity) of the aptamer sensor. Here, we present a systematic study of several approaches, including electrochemical interrogation parameters and biomolecular engineering of the aptamer sequence, to develop a sensor for the detection of aminoglycoside antibiotics. Specifically, through a combination of optimizing the electrochemical signal and engineering the parent 26-nucleotide RNA aptamer sequence to undergo larger conformation changes, we develop several improved sensors. These sensors exhibit binding affinities ranging from 220 nM to 42 μM, as much as a 100-fold improved limit of detection in comparison to previously reported sensors, and a variety of linear ranges including the therapeutic window for tobramycin. These data demonstrate that rational engineering of the aptamer structure to create large conformation changes upon target binding leads to improved sensor performance. We believe that the sensor design guidelines outlined here represent a general strategy for developing new aptamer folding-based electrochemical sensors.

  10. Downlink data multiplexer

    NASA Technical Reports Server (NTRS)

    Holland, S. Douglas (Inventor); Steele, Glen F. (Inventor); Romero, Denise M. (Inventor); Koudelka, Robert David (Inventor)

    2008-01-01

    A data multiplexer that accommodates both industry standard CCSDS data packets and bits streams and standard IEEE 1394 data is described. The multiplexer provides a statistical allotment of bandwidth to the channels in turn, preferably four, but expandable in increments of four up to sixteen. A microcontroller determines bandwidth requested by the plurality of channels, as well as the bandwidth available, and meters out the available bandwidth on a statistical basis employing flow control to the input channels.

  11. Ultra-high-sensitivity stable-isotope probing of rRNA by high-throughput sequencing of isopycnic centrifugation gradients.

    PubMed

    Aoyagi, Tomo; Hanada, Satoshi; Itoh, Hideomi; Sato, Yuya; Ogata, Atsushi; Friedrich, Michael W; Kikuchi, Yoshitomo; Hori, Tomoyuki

    2015-04-01

    Stable isotope probing (SIP) of rRNA directly identifies microorganisms assimilating an isotopically labelled substrate. High-throughput DNA sequencing is available for label screening at high resolution and high sensitivity, yet its effectiveness and validity remain to be clarified. Here, we investigated whether the detection sensitivity of rRNA-SIP could be improved by using Illumina sequencing in place of terminal restriction fragment length polymorphism (T-RFLP) analysis. A dilution series of (13) C-labelled RNA from Escherichia coli (1-0.0001%) and unlabelled RNA from Bacillus subtilis was density separated and fractionated. Illumina sequencing of isopycnic centrifugation gradients was able to detect (13) C-labelled RNA in the heaviest fraction with a buoyant density of 1.798 g ml(-1) even at the mixing ratio of 0.001%, whereas the detection ability of T-RFLP was not lower than 0.5%. Quantitative reverse transcription polymerase chain reaction of the density-separated RNAs showed that (13) C-labelled RNAs at mixing ratios of 0.05-0.001% had definitely accumulated in the heaviest fraction. Consequently, high-throughput sequencing provided up to 500-fold higher sensitivity for screening of (13) C-labelled RNA than T-RFLP. Ultra-high-sensitivity rRNA-SIP represents a clear advance towards a more complete understanding of microbial ecosystem function, including the ecophysiology of rare microorganisms in various natural environments.

  12. Sensitivity of hepatitis C virus RNA to the antiviral enzyme ribonuclease L is determined by a subset of efficient cleavage sites.

    PubMed

    Han, Jian-Qiu; Wroblewski, Gerggory; Xu, Zan; Silverman, Robert H; Barton, David J

    2004-11-01

    Ribonuclease L (RNase L) cleaves RNA predominantly at single-stranded UA and UU dinucleotides. Intriguingly, hepatitis C virus (HCV) RNAs have a paucity of UA and UU dinucleotides, and relatively interferon (IFN)-resistant strains have fewer UA and UU dinucleotides than do more IFN-sensitive strains. In this study, we found that contextual features of UA and UU dinucleotides dramatically affected the efficiency of RNase L cleavage in HCV RNA. HCV genotype la RNA was cleaved by RNase L into fragments 200-1000 bases in length, consistent with 10-50 RNase L cleavage sites within the 9650-base long viral RNA. Using primer extension, we found that HCV RNA structures with multiple single-stranded UA and UU dinucleotides were cleaved most efficiently by RNase L. UA and UU dinucleotides with 3' proximal C or G residues were cleaved infrequently, whereas UA and UU dinucleotides within dsRNA structures were not cleaved. 5'-GUAC-3' and 5'-CUUC-3' were particularly unfavorable contexts for cleavage by RNase L. More than 60% of the UA and UU dinucleotides in HCV la RNA were not cleaved by RNase L because of these contextual features. The 10-30 most efficiently cleaved sites were responsible for approximately 50%-85% of all RNase L cleavage events. Our data indicate that a relatively small number of the UA and UU dinucleotides in HCV RNA mediate the overall sensitivity of HCV RNA to cleavage by RNase L.

  13. Compact spatial multiplexers for mode division multiplexing.

    PubMed

    Chen, Haoshuo; van Uden, Roy; Okonkwo, Chigo; Koonen, Ton

    2014-12-29

    Spatial multiplexer (SMUX) for mode division multiplexing (MDM) has evolved from mode-selective excitation, multiple-spot and photonic-lantern based solutions in order to minimize both mode-dependent loss (MDL) and coupler insertion loss (CIL). This paper discusses the implementation of all the three solutions by compact components in a small footprint. Moreover, the compact SMUX can be manufactured in mass production and packaged to assure high reliability. First, push-pull scheme and center launch based SMUXes are demonstrated on two mostly-popular photonic integration platforms: Silicon-on-insulator (SOI) and Indium Phosphide (InP) for selectively exciting LP01 and LP11 modes. 2-dimensional (2D) top-coupling by using vertical emitters is explored to provide a coupling interface between a few-mode fiber (FMF) and the photonic integrated SMUX. SOI-based grating couplers and InP-based 45° vertical mirrors are proposed and researched as vertical emitters in each platform. Second, a 3-spot SMUX is realized on an InP-based circuit through employing 45° vertical mirrors. Third, as a newly-emerging photonic integration platform, laser-inscribed 3D waveguide (3DW) technology is applied for a fully-packaged dual-channel 6-mode SMUX including two 6-core photonic lantern structures as mode multiplexer and demultiplexer, respectively.

  14. Increased Expression of the dsRNA-Activated Protein Kinase PKR in Breast Cancer Promotes Sensitivity to Doxorubicin

    PubMed Central

    Bennett, Richard L.; Carruthers, Aubrey L.; Hui, Teng; Kerney, Krystal R.; Liu, Xiangfei; May, W. Stratford

    2012-01-01

    It has been reported that the expression and activity of the interferon-inducible, dsRNA-dependent protein kinase, PKR, is increased in mammary carcinoma cell lines and primary tumor samples. To extend these findings and determine how PKR signaling may affect breast cancer cell sensitivity to chemotherapy, we measured PKR expression by immunohistochemical staining of 538 cases of primary breast cancer and normal tissues. Significantly, PKR expression was elevated in ductal, lobular and squamous cell carcinomas or lymph node metastases but not in either benign tumor specimens or cases of inflammation compared to normal tissues. Furthermore, PKR expression was increased in precancerous stages of mammary cell hyperplasia and dysplasia compared to normal tissues, indicating that PKR expression may be upregulated by the process of tumorigenesis. To test the function of PKR in breast cancer, we generated MCF7, T-47D and MDA-MB-231 breast cancer cell lines with significantly reduced PKR expression by siRNA knockdown. Importantly, while knockdown of PKR expression had no effect on cell proliferation under normal growth conditions, MCF7, T-47D or MDA-MB-231 cells with reduced PKR expression or treated with a small molecule PKR inhibitor were significantly less sensitive to doxorubicin or H2O2-induced toxicity compared to control cells. In addition, the rate of eIF2α phosphorylation following treatment with doxorubicin was delayed in breast cancer cell lines with decreased PKR expression. Significantly, treatment of breast cancer lines with reduced PKR expression with either interferon-α, which increases PKR expression, or salubrinal, which increases eIF2α phosphorylation, restored doxorubicin sensitivity to normal levels. Taken together these results indicate that increased PKR expression in primary breast cancer tissues may serve as a biomarker for response to doxorubicin-containing chemotherapy and that future therapeutic approaches to promote PKR expression

  15. An MRPS12 mutation modifies aminoglycoside sensitivity caused by 12S rRNA mutations

    PubMed Central

    Emperador, Sonia; Pacheu-Grau, David; Bayona-Bafaluy, M. Pilar; Garrido-Pérez, Nuria; Martín-Navarro, Antonio; López-Pérez, Manuel J.; Montoya, Julio; Ruiz-Pesini, Eduardo

    2015-01-01

    Several homoplasmic pathologic mutations in mitochondrial DNA, such as those causing Leber hereditary optic neuropathy or non-syndromic hearing loss, show incomplete penetrance. Therefore, other elements must modify their pathogenicity. Discovery of these modifying factors is not an easy task because in multifactorial diseases conventional genetic approaches may not always be informative. Here, we have taken an evolutionary approach to unmask putative modifying factors for a particular homoplasmic pathologic mutation causing aminoglycoside-induced and non-syndromic hearing loss, the m.1494C>T transition in the mitochondrial DNA. The mutation is located in the decoding site of the mitochondrial ribosomal RNA. We first looked at mammalian species that had fixed the human pathologic mutation. These mutations are called compensated pathogenic deviations because an organism carrying one must also have another that suppresses the deleterious effect of the first. We found that species from the primate family Cercopithecidae (old world monkeys) harbor the m.1494T allele even if their auditory function is normal. In humans the m.1494T allele increases the susceptibility to aminoglycosides. However, in primary fibroblasts from a Cercopithecidae species, aminoglycosides do not impair cell growth, respiratory complex IV activity and quantity or the mitochondrial protein synthesis. Interestingly, this species also carries a fixed mutation in the mitochondrial ribosomal protein S12. We show that the expression of this variant in a human m.1494T cell line reduces its susceptibility to aminoglycosides. Because several mutations in this human protein have been described, they may possibly explain the absence of pathologic phenotype in some pedigree members with the most frequent pathologic mutations in mitochondrial ribosomal RNA. PMID:25642242

  16. CuO-induced signal amplification strategy for multiplexed photoelectrochemical immunosensing using CdS sensitized ZnO nanotubes arrays as photoactive material and AuPd alloy nanoparticles as electron sink.

    PubMed

    Sun, Guoqiang; Zhang, Yan; Kong, Qingkun; Zheng, Xiaoxiao; Yu, Jinghua; Song, Xianrang

    2015-04-15

    In this work, multiplexed photoelectrochemical (PEC) immunoassays are introduced into an indium tin oxide (ITO) device. Firstly, the ITO device is fabricated using a simple acid etch treatment method. Secondly, AuPd alloy nanoparticles are electro-deposited on ITO working electrodes as electron sink to construct the immunosensor platform. After that, ZnO nanotubes (ZNTs) arrays are synthesized via chemical etching of ZnO nanorods that are grown on AuPd surface by electrochemical deposition method. Subsequently, CdS is electro-deposited on ZNTs arrays and used as photoactive material. Then, CuO nanoseeds are labeled with signal antibodies and firstly used as PEC signal amplification label. The introduction of CuO brings signal amplification because of the conduction band (CB) of both CuO and ZnO are lower than that of CdS, CuO will compete the photo-induced electrons in CB of CdS with ZnO, leading to the decrease of the photocurrent intensity. Using cancer antigen 125, prostate specific antigen and α-fetoprotein as model analytes, the proposed immunoassay exhibits excellent precision and sensitivity. Meanwhile, this work provides a promising, addressable and simple strategy for the multi-detection of tumor markers.

  17. The intranuclear mobility of messenger RNA binding proteins is ATP dependent and temperature sensitive.

    PubMed

    Calapez, Alexandre; Pereira, Henrique M; Calado, Angelo; Braga, José; Rino, José; Carvalho, Célia; Tavanez, João Paulo; Wahle, Elmar; Rosa, Agostinho C; Carmo-Fonseca, Maria

    2002-12-01

    After being released from transcription sites, messenger ribonucleoprotein particles (mRNPs) must reach the nuclear pore complexes in order to be translocated to the cytoplasm. Whether the intranuclear movement of mRNPs results largely from Brownian motion or involves molecular motors remains unknown. Here we have used quantitative photobleaching techniques to monitor the intranuclear mobility of protein components of mRNPs tagged with GFP. The results show that the diffusion coefficients of the poly(A)-binding protein II (PABP2) and the export factor TAP are significantly reduced when these proteins are bound to mRNP complexes, as compared with nonbound proteins. The data further show that the mobility of wild-type PABP2 and TAP, but not of a point mutant variant of PABP2 that fails to bind to RNA, is significantly reduced when cells are ATP depleted or incubated at 22 degrees C. Energy depletion has only minor effects on the intranuclear mobility of a 2,000-kD dextran (which corresponds approximately in size to 40S mRNP particles), suggesting that the reduced mobility of PABP2 and TAP is not caused by a general alteration of the nuclear environment. Taken together, the data suggest that the mobility of mRNPs in the living cell nucleus involves a combination of passive diffusion and ATP-dependent processes.

  18. The intranuclear mobility of messenger RNA binding proteins is ATP dependent and temperature sensitive

    PubMed Central

    Calapez, Alexandre; Pereira, Henrique M.; Calado, Angelo; Braga, José; Rino, José; Carvalho, Célia; Tavanez, João Paulo; Wahle, Elmar; Rosa, Agostinho C.; Carmo-Fonseca, Maria

    2002-01-01

    fAter being released from transcription sites, messenger ribonucleoprotein particles (mRNPs) must reach the nuclear pore complexes in order to be translocated to the cytoplasm. Whether the intranuclear movement of mRNPs results largely from Brownian motion or involves molecular motors remains unknown. Here we have used quantitative photobleaching techniques to monitor the intranuclear mobility of protein components of mRNPs tagged with GFP. The results show that the diffusion coefficients of the poly(A)-binding protein II (PABP2) and the export factor TAP are significantly reduced when these proteins are bound to mRNP complexes, as compared with nonbound proteins. The data further show that the mobility of wild-type PABP2 and TAP, but not of a point mutant variant of PABP2 that fails to bind to RNA, is significantly reduced when cells are ATP depleted or incubated at 22°C. Energy depletion has only minor effects on the intranuclear mobility of a 2,000-kD dextran (which corresponds approximately in size to 40S mRNP particles), suggesting that the reduced mobility of PABP2 and TAP is not caused by a general alteration of the nuclear environment. Taken together, the data suggest that the mobility of mRNPs in the living cell nucleus involves a combination of passive diffusion and ATP-dependent processes. PMID:12473688

  19. MicroRNA-16 sensitizes breast cancer cells to paclitaxel through suppression of IKBKB expression

    PubMed Central

    Tang, Xueyuan; Jin, Long; Cao, Peiguo; Cao, Ke; Huang, Chenghui; Luo, Yanwei; Ma, Jian; Shen, Shourong; Tan, Ming; Li, Xiayu; Zhou, Ming

    2016-01-01

    Paclitaxel (Taxol) is an effective chemotherapeutic agent for treating breast cancer patients. However, chemoresistance is a major obstacle in cancer treatment. Here, we showed that overexpression of miR-16 promoted Taxol-induced cytotoxicity and apoptosis in breast cancer cells. Furthermore, IκB kinase β (IKBKB) was identified as a direct target of miR-16. Up-regulation of IKBKB suppressed Taxol-induced apoptosis and led to an increased resistance to Taxol, and restoring IKBKB expression in miR-16-overexpressing breast cancer cells recovered Taxol resistance. Moreover, miR-16 was highly expressed in Taxol-sensitive breast cancer tissues compared with Taxol-resistant tissues, and there was an inverse correlation between miR-16 expression and IKBKB expression in breast cancer tissues. The expression levels of miR-16 were negatively associated with T stages, whereas the expression of IKBKB was positively correlated with T stages, lymph node metastasis and clinical stages. Taken together, our data demonstrates that miR-16 sensitizes breast cancer cells to Taxol through the suppression of IKBKB expression, and targeting miR-16/IKBKB axis will be a promising strategy for overcoming Taxol resistance in breast cancer. PMID:26993770

  20. Effect of Alpha-Hederin, the active constituent of Nigella sativa, on miRNA-126, IL-13 mRNA levels and inflammation of lungs in ovalbumin-sensitized male rats

    PubMed Central

    Fallahi, Maryam; Keyhanmanesh, Rana; Khamaneh, Amir Mahdi; Ebrahimi Saadatlou, Mohammad Ali; Saadat, Saeideh; Ebrahimi, Hadi

    2016-01-01

    Objective: In previous studies the therapeutic effects of Nigella sativa have been demonstrated on asthmatic animals. In the present study, the preventive effect of single dose of alpha-hederin, its active constituent, has been evaluated on lung inflammation and some inflammatory mediators in lungs of ovalbumin sensitized rat in order to elicit its mechanism. Materials and Methods: Forty rats were randomly grouped in 4 groups; control (C), sensitized (S), sensitized pretreated groups with thymoquinone (3 mg/kg i.p., S+TQ) and alpha-hederin (0.02 mg/kg i.p., S+AH). Levels of IL-13 mRNA and miRNA-126 in lung tissue and its pathological changes in each group were assessed. Results: Elevated levels of miRNA-126, IL-13 mRNA and pathological changes were observed in the sensitized group compared to the control group (p<0.001 to p<0.05). All of these factors were significantly reduced in S+TQ and S+AH groups in comparison to S group (p<0.001 to p<0.05). Although alpha-hederin decreased the levels of miRNA-126, IL-13 mRNA and pathological changes in comparison with thymoquinone, the results were statistically not significant. Conclusion: The results suggested that alpha-hederin had preventive effect on sensitized rats like thymoquinone. It may intervene in miRNA-126 expression, which consequently could interfere with IL-13 secretion pathway leading to a reduction in inflammatory responses. PMID:27247924

  1. Multiplexed Biosensors for Mycotoxins.

    PubMed

    Maragos, Chris M

    2016-07-01

    Significant progress has been made in the development of biosensors that can be used to detect low-MW toxins produced by fungi (mycotoxins). The number of formats that have been investigated is impressive and is an indication of the importance attached to finding easy-to-use, accurate, and rapid methods for detecting these toxins in commodities and foods. This review explores the details of multiplexed biosensors based on many formats, including multiplexed immunoassays, suspension arrays, membrane-based devices (flow-through and immunochromatographic), and planar microarrays. Each assay format has its own strengths and areas that need improvement. Certain formats, such as multiplexed immunochromatographic devices, are well developed and relatively easy to use, and in some cases, commercial products are being sold. Others, such as the suspension arrays and microarrays, are laboratory-based assays that, although more complicated, are also more amenable to a larger scale of multiplexing. The diversity of such efforts and the multitude of formats under investigation suggest that multiple solutions will be found to satisfy the need for multiplexed toxin detection. PMID:27455928

  2. Simultaneous delivery of Paclitaxel and Bcl-2 siRNA via pH-Sensitive liposomal nanocarrier for the synergistic treatment of melanoma

    PubMed Central

    Reddy, Teegala Lakshminarayan; Garikapati, Koteswara Rao; Reddy, S. Gopal; Reddy, B. V. Subba; Yadav, J. S.; Bhadra, Utpal; Bhadra, Manika Pal

    2016-01-01

    pH-sensitive drug carriers that are sensitive to the acidic (pH = ~6.5) microenvironments of tumor tissues have been primarily used as effective drug/gene/siRNA/microRNA carriers for releasing their payloads to tumor cells/tissues. Resistance to various drugs has become a big hurdle in systemic chemotherapy in cancer. Therefore delivery of chemotherapeutic agents and siRNA’s targeting anti apoptotic genes possess advantages to overcome the efflux pump mediated and anti apoptosis-related drug resistance. Here, we report the development of nanocarrier system prepared from kojic acid backbone-based cationic amphiphile containing endosomal pH-sensitive imidazole ring. This pH-sensitive liposomal nanocarrier effectively delivers anti-cancer drug (Paclitaxel; PTX) and siRNA (Bcl-2), and significantly inhibits cell proliferation and reduces tumor growth. Tumor inhibition response attributes to the synergistic effect of PTX potency and MDR reversing ability of Bcl-2 siRNA in the tumor supporting that kojic acid based liposomal pH-sensitive nanocarrier as efficient vehicle for systemic co-delivery of drugs and siRNA. PMID:27786239

  3. MicroRNA-328 directly targets p21-activated protein kinase 6 inhibiting prostate cancer proliferation and enhancing docetaxel sensitivity

    PubMed Central

    LIU, CHUNHUI; ZHANG, LEI; HUANG, YEQING; LU, KAI; TAO, TAO; CHEN, SHUQIU; ZHANG, XIAOWEN; GUAN, HAN; CHEN, MING; XU, BIN

    2015-01-01

    Prostate cancer (Pca) has one of the highest mortality rates for malignant cancers worldwide. Previous research has demonstrated that numerous genes are aberrantly expressed during Pca onset and development. p21-activated protein kinase 6 (PAK6) is known to be overexpressed in primary and metastatic Pca, however the mechanism of this aberrant expression remains unknown. In the present study, immunohistochemistry demonstrated that PAK6 is overexpressed in castration-resistant Pca (CRPC). Furthermore, PAK6 overexpression was regulated by microRNA (miR)-328. Luciferase reporter assay and western blot analysis indicated that PAK6 was directly targeted by miR-328. Forced expression of miR-328 enhanced docetaxel sensitivity, inhibited cell proliferation and promoted cell apoptosis without affecting the cell cycle. This indicates that miR-328 performs important functions in CRPC progression via PAK6 regulation. This mechanism may be used to enhance the effect of docetaxel. PMID:26459798

  4. Microwave multiplex readout for superconducting sensors

    NASA Astrophysics Data System (ADS)

    Ferri, E.; Becker, D.; Bennett, D.; Faverzani, M.; Fowler, J.; Gard, J.; Giachero, A.; Hays-Wehle, J.; Hilton, G.; Maino, M.; Mates, J.; Puiu, A.; Nucciotti, A.; Reintsema, C.; Schmidt, D.; Swetz, D.; Ullom, J.; Vale, L.

    2016-07-01

    The absolute neutrino mass scale is still an outstanding challenge in both particle physics and cosmology. The calorimetric measurement of the energy released in a nuclear beta decay is a powerful tool to determine the effective electron-neutrino mass. In the last years, the progress on low temperature detector technologies has allowed to design large scale experiments aiming at pushing down the sensitivity on the neutrino mass below 1 eV. Even with outstanding performances in both energy (~ eV on keV) and time resolution (~ 1 μs) on the single channel, a large number of detectors working in parallel is required to reach a sub-eV sensitivity. Microwave frequency domain readout is the best available technique to readout large array of low temperature detectors, such as Transition Edge Sensors (TESs) or Microwave Kinetic Inductance Detectors (MKIDs). In this way a multiplex factor of the order of thousands can be reached, limited only by the bandwidth of the available commercial fast digitizers. This microwave multiplexing system will be used to readout the HOLMES detectors, an array of 1000 microcalorimeters based on TES sensors in which the 163Ho will be implanted. HOLMES is a new experiment for measuring the electron neutrino mass by means of the electron capture (EC) decay of 163Ho. We present here the microwave frequency multiplex which will be used in the HOLMES experiment and the microwave frequency multiplex used to readout the MKID detectors developed in Milan as well.

  5. Inhibition of microRNA-155 sensitizes lung cancer cells to irradiation via suppression of HK2-modulated glucose metabolism.

    PubMed

    Lv, Xin; Yao, Li; Zhang, Jianli; Han, Ping; Li, Cuiyun

    2016-08-01

    MicroRNAs (miRNAs) are small non-coding regulatory RNAs, which are involved in the post-transcriptional regulation of gene expression. miRNA (miR)-155, which has previously been reported to be overexpressed in lung cancer, is correlated with poor patient prognosis. The present study aimed to investigate the effects of miR‑155 on the radiosensitivity of human non‑small cell lung cancer (NSCLC) cells. To explore the roles of miRNAs in the regulation of irradiation sensitivity of human lung cancer cells, the expressions of miR‑155 in response to irradiation, have been studied by RT‑qPCR, and the putative direct target of miR‑155 was identified by western blot and luciferase assays. The results of the present study revealed that the expression of miR‑155 was induced by irradiation, thus suggesting a positive correlation between miR‑155 and radiosensitivity. Furthermore, overexpression of miR‑155 rendered lung cancer cells resistant to irradiation. In addition, hexokinase 2 (HK2) was identified as an indirect target of miR‑155; exogenous overexpression of miR‑155 upregulated the expression of HK2, whereas inhibition of miR‑155 by antisense miRNA suppressed HK2 expression. In addition, HK2‑modulated glucose metabolism was significantly upregulated by overexpression of miR‑155. Notably, inhibition of miR‑155 sensitized lung cancer cells to irradiation via suppression of glucose metabolism. In conclusion, the present study reported a novel function for miR‑155 in the regulation of NSCLC cell radiosensitivity, thus suggesting that miR‑155 may be considered a therapeutic target for the development of anticancer drugs. PMID:27315591

  6. Antagonism of miRNA-21 Sensitizes Human Gastric Cancer Cells to Paclitaxel.

    PubMed

    Jin, Bo; Liu, Yanping; Wang, Haijiang

    2015-05-01

    The development of drug resistance has largely limited the clinical outcome of anti-cancer treatment. Recent work has highlighted the involvement of non-coding RNAs, microRNAs (miRNAs), in cancer development. The present study aimed to investigate the role of miR-21 in the development of drug resistance to paclitaxel in gastric cancer cells. Our study found that the expression of miR-21 upregulated in the paclitaxel resistant cell line SGC7901/paclitaxel compared to its parental line SGC7901. Moreover, over-expression of miR-21 significantly decreased antiproliferative effects and apoptosis induced by paclitaxel, while knockdown of miR-21 dramatically increased antiproliferative effects and apoptosis induction by paclitaxel. Moreover, our results demonstrated that miR-21 may modulate the sensitivity to PTX, at least in part, by regulating the expression of P-glycoprotein. PMID:27040946

  7. Micro RNA 100 sensitizes luminal A breast cancer cells to paclitaxel treatment in part by targeting mTOR

    PubMed Central

    He, Yuan; Fu, Xing; Fu, Liya; Zhu, Zhengmao; Fu, Li; Dong, Jin-Tang

    2016-01-01

    Luminal A breast cancer usually responds to hormonal therapies but does not benefit from chemotherapies, including microtubule-targeted paclitaxel. MicroRNAs could play a role in mediating this differential response. In this study, we examined the role of micro RNA 100 (miR-100) in the sensitivity of breast cancer to paclitaxel treatment. We found that while miR-100 was downregulated in both human breast cancer primary tumors and cell lines, the degree of downregulation was greater in the luminal A subtype than in other subtypes. The IC50 of paclitaxel was much higher in luminal A than in basal-like breast cancer cell lines. Ectopic miR-100 expression in the MCF-7 luminal A cell line enhanced the effect of paclitaxel on cell cycle arrest, multinucleation, and apoptosis, while knockdown of miR-100 in the MDA-MB-231 basal-like line compromised these effects. Similarly, overexpression of miR-100 enhanced the effects of paclitaxel on tumorigenesis in MCF-7 cells. Rapamycin-mediated inhibition of the mammalian target of rapamycin (mTOR), a target of miR-100, also sensitized MCF-7 cells to paclitaxel. Gene set enrichment analysis showed that genes that are part of the known paclitaxel-sensitive signature had a significant expression correlation with miR-100 in breast cancer samples. In addition, patients with lower levels of miR-100 expression had worse overall survival. These results suggest that miR-100 plays a causal role in determining the sensitivity of breast cancers to paclitaxel treatment. PMID:26744318

  8. Increased Sensitivity to Chemotherapy Induced by CpG-ODN Treatment Is Mediated by microRNA Modulation

    PubMed Central

    Sommariva, Michele; Cataldo, Alessandra; Canevari, Silvana; Mezzanzanica, Delia; Iorio, Marilena V.; Tagliabue, Elda; Balsari, Andrea

    2013-01-01

    We recently reported that peritumoral CpG-ODN treatment, activating TLR-9 expressing cells in tumor microenvironment, induces modulation of genes involved in DNA repair and sensitizes cancer cells to DNA-damaging cisplatin treatment. Here, we investigated whether this treatment induces modulation of miRNAs in tumor cells and their relevance to chemotherapy response. Array analysis identified 20 differentially expressed miRNAs in human IGROV-1 ovarian tumor cells from CpG-ODN-treated mice versus controls (16 down- and 4 up-regulated). Evaluation of the role of the 3 most differentially expressed miRNAs on sensitivity to cisplatin of IGROV-1 cells revealed significantly increased cisplatin cytotoxicity upon ectopic expression of hsa-miR-302b (up-modulated in our array), but no increased effect upon reduced expression of hsa-miR-424 or hsa-miR-340 (down-modulated in our array). Accordingly, hsa-miR-302b expression was significantly associated with time to relapse or overall survival in two data sets of platinum-treated ovarian cancer patients. Use of bio-informatics tools identified 19 mRNAs potentially targeted by hsa-miR-302b, including HDAC4 gene, which has been reported to mediate cisplatin sensitivity in ovarian cancer. Both HDAC4 mRNA and protein levels were significantly reduced in IGROV-1 cells overexpressing hsa-miR-302b. Altogether, these findings indicate that hsa-miR-302b acts as a “chemosensitizer” in human ovarian carcinoma cells and may represent a biomarker able to predict response to cisplatin treatment. Moreover, the identification of miRNAs that improve sensitivity to chemotherapy provides the experimental underpinning for their possible future clinical use. PMID:23484053

  9. MicroRNA-566 activates EGFR signaling and its inhibition sensitizes glioblastoma cells to nimotuzumab

    PubMed Central

    2014-01-01

    Background Epidermal growth factor receptor (EGFR) is amplified in 40% of human glioblastomas. However, most glioblastoma patients respond poorly to anti-EGFR therapy. MicroRNAs can function as either oncogenes or tumor suppressor genes, and have been shown to play an important role in cancer cell proliferation, invasion and apoptosis. Whether microRNAs can impact the therapeutic effects of EGFR inhibitors in glioblastoma is unknown. Methods miR-566 expression levels were detected in glioma cell lines, using real-time quantitative RT-PCR (qRT-PCR). Luciferase reporter assays and Western blots were used to validate VHL as a direct target gene of miR-566. Cell proliferation, invasion, cell cycle distribution and apoptosis were also examined to confirm whether miR-566 inhibition could sensitize anti-EGFR therapy. Results In this study, we demonstrated that miR-566 is up-regulated in human glioma cell lines and inhibition of miR-566 decreased the activity of the EGFR pathway. Lentiviral mediated inhibition of miR-566 in glioblastoma cell lines significantly inhibited cell proliferation and invasion and led to cell cycle arrest in the G0/G1 phase. In addition, we identified von Hippel-Lindau (VHL) as a novel functional target of miR-566. VHL regulates the formation of the β-catenin/hypoxia-inducible factors-1α complex under miR-566 regulation. Conclusions miR-566 activated EGFR signaling and its inhibition sensitized glioblastoma cells to anti-EGFR therapy. PMID:24650032

  10. Multiplex nested reverse transcription-polymerase chain reaction for respiratory viruses in acute otitis media.

    PubMed

    Ishibashi, Toshio; Monobe, Hiroko; Nomura, Yuka; Shinogami, Masanobu; Yano, Jun

    2003-03-01

    Because respiratory viruses play an important role in the causation and pathogenesis of acute otitis media (AOM), determining which virus has infected a child is important with respect to vaccines and antiviral drugs. In some instances, this information might be used to prevent the occurrence of AOM. We used a rapid, economical, and sensitive diagnostic system involving a multiplex nested reverse transcription-polymerase chain reaction (RT-PCR) assay to detect various respiratory viruses in clinical specimens of middle ear fluid (MEF) from children with AOM in our hospital. Multiplex RT-PCR was completed on 40 MEF samples from 28 infants and children less than 6 years old with AOM. Viral RNA was detected in 17 MEF samples (43%). Respiratory syncytial virus type A was present in 12 samples, adenovirus in 3, rhinovirus in 2, and influenza A (H3N2) in 1. The multiplex RT-PCR assay is recommended to clinical laboratories that are considering adoption of a molecular technique for viral diagnosis.

  11. A multiplexed quantum memory.

    PubMed

    Lan, S-Y; Radnaev, A G; Collins, O A; Matsukevich, D N; Kennedy, T A; Kuzmich, A

    2009-08-01

    A quantum repeater is a system for long-distance quantum communication that employs quantum memory elements to mitigate optical fiber transmission losses. The multiplexed quantum memory (O. A. Collins, S. D. Jenkins, A. Kuzmich, and T. A. B. Kennedy, Phys. Rev. Lett. 98, 060502 (2007)) has been shown theoretically to reduce quantum memory time requirements. We present an initial implementation of a multiplexed quantum memory element in a cold rubidium gas. We show that it is possible to create atomic excitations in arbitrary memory element pairs and demonstrate the violation of Bell's inequality for light fields generated during the write and read processes.

  12. A nucleic acid probe labeled with desmethyl thiazole orange: a new type of hybridization-sensitive fluorescent oligonucleotide for live-cell RNA imaging.

    PubMed

    Okamoto, Akimitsu; Sugizaki, Kaori; Yuki, Mizue; Yanagisawa, Hiroyuki; Ikeda, Shuji; Sueoka, Takuma; Hayashi, Gosuke; Wang, Dan Ohtan

    2013-01-14

    A new fluorescent nucleotide with desmethyl thiazole orange dyes, D'(505), has been developed for expansion of the function of fluorescent probes for live-cell RNA imaging. The nucleoside unit of D'(505) for DNA autosynthesis was soluble in organic solvents, which made the preparation of nucleoside units and the reactions in the cycles of DNA synthesis more efficient. The dyes of D'(505)-containing oligodeoxynucleotide were protonated below pH 7 and the oligodeoxynucleotide exhibited hybridization-sensitive fluorescence emission through the control of excitonic interactions of the dyes of D'(505). The simplified procedure and effective hybridization-sensitive fluorescence emission produced multicolored hybridization-sensitive fluorescent probes, which were useful for live-cell RNA imaging. The acceptor-bleaching method gave us information on RNA in a specific cell among many living cells.

  13. Color-Multiplexing-Based Fluorescent Test Paper: Dosage-Sensitive Visualization of Arsenic(III) with Discernable Scale as Low as 5 ppb.

    PubMed

    Zhou, Yujie; Huang, Xiaoyan; Liu, Cui; Zhang, Ruilong; Gu, Xiaoling; Guan, Guijian; Jiang, Changlong; Zhang, Liying; Du, Shuhu; Liu, Bianhua; Han, Ming-Yong; Zhang, Zhongping

    2016-06-21

    Fluorescent colorimetry test papers are promising for the assays of environments, medicines, and foods by the observation of the naked eye on the variations of fluorescence brightness and color. Unlike dye-absorption-based pH test paper, however, the fluorescent test papers with wide color-emissive variations with target dosages for accurate quantification remain unsuccessful even if the multicolorful fluorescent probes are used. Here, we report the dosage-sensitive fluorescent colorimetry test paper with a very wide/consecutive "from red to cyan" response to the presence and amount of arsenic ions, As(III). Red quantum dots (QDs) were modified with glutathione and dithiothreitol to obtain the supersensitivity to As(III) by the quenching of red fluorescence through the formation of dispersive QDs aggregates. A small amount of cyan carbon dots (CDs) with spectral blue-green components as the photostable internal standard were mixed into the QDs solution to produce a composited red fluorescence. Upon the addition of As(III) into the sensory solution, the fluorescence color could gradually be reversed from red to cyan with a detection limit of 1.7 ppb As(III). When the sensory solution was printed onto a piece of filter paper, surprisingly a serial of color evolution from peach to pink to orange to khaki to yellowish to yellow-green to final cyan with the addition of As(III) was displayed and clearly discerned the dosage scale as low as 5 ppb. The methodology reported here opens a novel pathway toward the real applications of fluorescent test papers. PMID:27230307

  14. Matrix metalloproteinase 2-sensitive multifunctional polymeric micelles for tumor-specific co-delivery of siRNA and hydrophobic drugs.

    PubMed

    Zhu, Lin; Perche, Federico; Wang, Tao; Torchilin, Vladimir P

    2014-04-01

    Co-delivery of hydrophilic siRNA and hydrophobic drugs is one of the major challenges for nanomaterial-based medicine. Here, we present a simple but multifunctional micellar platform constructed by a matrix metalloproteinase 2 (MMP2)-sensitive copolymer (PEG-pp-PEI-PE) via self-assembly for tumor-targeted siRNA and drug co-delivery. The micellar nanocarrier possesses several key features for siRNA and drug delivery, including (i) excellent stability; (ii) efficient siRNA condensation by PEI; (iii) hydrophobic drug solubilization in the lipid "core"; (iv) passive tumor targeting via the enhanced permeability and retention (EPR) effect; (v) tumor targeting triggered by the up-regulated tumoral MMP2; and (vi) enhanced cell internalization after MMP2-activated exposure of the previously hidden PEI. These cooperative functions ensure the improved tumor targetability, enhanced tumor cell internalization, and synergistic antitumor activity of co-loaded siRNA and drug. PMID:24529391

  15. Biosensor-based microRNA detection: techniques, design, performance, and challenges.

    PubMed

    Johnson, Blake N; Mutharasan, Raj

    2014-04-01

    The current state of biosensor-based techniques for amplification-free microRNA (miRNA) detection is critically reviewed. Comparison with non-sensor and amplification-based molecular techniques (MTs), such as polymerase-based methods, is made in terms of transduction mechanism, associated protocol, and sensitivity. Challenges associated with miRNA hybridization thermodynamics which affect assay selectivity and amplification bias are briefly discussed. Electrochemical, electromechanical, and optical classes of miRNA biosensors are reviewed in terms of transduction mechanism, limit of detection (LOD), time-to-results (TTR), multiplexing potential, and measurement robustness. Current trends suggest that biosensor-based techniques (BTs) for miRNA assay will complement MTs due to the advantages of amplification-free detection, LOD being femtomolar (fM)-attomolar (aM), short TTR, multiplexing capability, and minimal sample preparation requirement. Areas of future importance in miRNA BT development are presented which include focus on achieving high measurement confidence and multiplexing capabilities.

  16. Differential Sensitivity of Bat Cells to Infection by Enveloped RNA Viruses: Coronaviruses, Paramyxoviruses, Filoviruses, and Influenza Viruses

    PubMed Central

    Hoffmann, Markus; Müller, Marcel Alexander; Drexler, Jan Felix; Glende, Jörg; Erdt, Meike; Gützkow, Tim; Losemann, Christoph; Binger, Tabea; Deng, Hongkui; Schwegmann-Weßels, Christel; Esser, Karl-Heinz; Drosten, Christian; Herrler, Georg

    2013-01-01

    Bats (Chiroptera) host major human pathogenic viruses including corona-, paramyxo, rhabdo- and filoviruses. We analyzed six different cell lines from either Yinpterochiroptera (including African flying foxes and a rhinolophid bat) or Yangochiroptera (genera Carollia and Tadarida) for susceptibility to infection by different enveloped RNA viruses. None of the cells were sensitive to infection by transmissible gastroenteritis virus (TGEV), a porcine coronavirus, or to infection mediated by the Spike (S) protein of SARS-coronavirus (SARS-CoV) incorporated into pseudotypes based on vesicular stomatitis virus (VSV). The resistance to infection was overcome if cells were transfected to express the respective cellular receptor, porcine aminopeptidase N for TGEV or angiotensin-converting enzyme 2 for SARS-CoV. VSV pseudotypes containing the S proteins of two bat SARS-related CoV (Bg08 and Rp3) were unable to infect any of the six tested bat cell lines. By contrast, viral pseudotypes containing the surface protein GP of Marburg virus from the family Filoviridae infected all six cell lines though at different efficiency. Notably, all cells were sensitive to infection by two paramyxoviruses (Sendai virus and bovine respiratory syncytial virus) and three influenza viruses from different subtypes. These results indicate that bat cells are more resistant to infection by coronaviruses than to infection by paramyxoviruses, filoviruses and influenza viruses. Furthermore, these results show a receptor-dependent restriction of the infection of bat cells by CoV. The implications for the isolation of coronaviruses from bats are discussed. PMID:24023659

  17. Differential sensitivity of bat cells to infection by enveloped RNA viruses: coronaviruses, paramyxoviruses, filoviruses, and influenza viruses.

    PubMed

    Hoffmann, Markus; Müller, Marcel Alexander; Drexler, Jan Felix; Glende, Jörg; Erdt, Meike; Gützkow, Tim; Losemann, Christoph; Binger, Tabea; Deng, Hongkui; Schwegmann-Weßels, Christel; Esser, Karl-Heinz; Drosten, Christian; Herrler, Georg

    2013-01-01

    Bats (Chiroptera) host major human pathogenic viruses including corona-, paramyxo, rhabdo- and filoviruses. We analyzed six different cell lines from either Yinpterochiroptera (including African flying foxes and a rhinolophid bat) or Yangochiroptera (genera Carollia and Tadarida) for susceptibility to infection by different enveloped RNA viruses. None of the cells were sensitive to infection by transmissible gastroenteritis virus (TGEV), a porcine coronavirus, or to infection mediated by the Spike (S) protein of SARS-coronavirus (SARS-CoV) incorporated into pseudotypes based on vesicular stomatitis virus (VSV). The resistance to infection was overcome if cells were transfected to express the respective cellular receptor, porcine aminopeptidase N for TGEV or angiotensin-converting enzyme 2 for SARS-CoV. VSV pseudotypes containing the S proteins of two bat SARS-related CoV (Bg08 and Rp3) were unable to infect any of the six tested bat cell lines. By contrast, viral pseudotypes containing the surface protein GP of Marburg virus from the family Filoviridae infected all six cell lines though at different efficiency. Notably, all cells were sensitive to infection by two paramyxoviruses (Sendai virus and bovine respiratory syncytial virus) and three influenza viruses from different subtypes. These results indicate that bat cells are more resistant to infection by coronaviruses than to infection by paramyxoviruses, filoviruses and influenza viruses. Furthermore, these results show a receptor-dependent restriction of the infection of bat cells by CoV. The implications for the isolation of coronaviruses from bats are discussed. PMID:24023659

  18. Acid-Sensitive Sheddable PEGylated PLGA Nanoparticles Increase the Delivery of TNF-α siRNA in Chronic Inflammation Sites.

    PubMed

    Aldayel, Abdulaziz M; Naguib, Youssef W; O'Mary, Hannah L; Li, Xu; Niu, Mengmeng; Ruwona, Tinashe B; Cui, Zhengrong

    2016-01-01

    There has been growing interest in utilizing small interfering RNA (siRNA) specific to pro-inflammatory cytokines, such as tumor necrosis factor-α ( TNF-α), in chronic inflammation therapy. However, delivery systems that can increase the distribution of the siRNA in chronic inflammation sites after intravenous administration are needed. Herein we report that innovative functionalization of the surface of siRNA-incorporated poly (lactic-co-glycolic) acid (PLGA) nanoparticles significantly increases the delivery of the siRNA in the chronic inflammation sites in a mouse model. The TNF-α siRNA incorporated PLGA nanoparticles were prepared by the standard double emulsion method, but using stearoyl-hydrazone-polyethylene glycol 2000, a unique acid-sensitive surface active agent, as the emulsifying agent, which renders (i) the nanoparticles PEGylated and (ii) the PEGylation sheddable in low pH environment such as that in chronic inflammation sites. In a mouse model of lipopolysaccharide-induced chronic inflammation, the acid-sensitive sheddable PEGylated PLGA nanoparticles showed significantly higher accumulation or distribution in chronic inflammation sites than PLGA nanoparticles prepared with an acid-insensitive emulsifying agent (i.e., stearoyl-amide-polyethylene glycol 2000) and significantly increased the distribution of the TNF-α siRNA incorporated into the nanoparticles in inflamed mouse foot. PMID:27434685

  19. MicroRNA-183 correlates cancer prognosis, regulates cancer proliferation and bufalin sensitivity in epithelial ovarian caner.

    PubMed

    Chen, Huixiao; Zhang, Ling; Zhang, Lili; Du, Jing; Wang, Hongying; Wang, Bin

    2016-01-01

    Background we intended to explore the functional implication of microRNA-183 (miR-183) in predicting clinical prognosis and regulating cancer proliferation and bufalin sensitivity in epithelial ovarian cancer (EOC). Methods In 75 EOC patients, miR-183 expression was examined, by quantitative RT-PCR (qRT-PCR), between paired EOC tumors and adjacent normal tissues, and between tumor samples from patients at early clinical stages and those at advanced clinical stages. The association of serum miR-183 and patients' clinicopathological variables were examined. The overall survival (OS) was estimated by Kaplan-Meier model. And the possibility of miR-183 as a prognostic biomarker for EOC was examined by cox proportional hazard regression model. In EOC cell lines SKOV3 and ES-2 cells, lentiviral transduction was conducted to genetically suppress miR-183. The effect of miR-183 downregulation on EOC in vitro growth, bufalin sensitivity and in vivo tumorigenicity were examined. Results MiR-183 was highly expressed in EOC tumors, as well ass in patients at advanced clinical stages. Serum miR-183 was significantly associated with major clinicopathological variables in EOC patients, such as clinical stage and lymph node metastases. High level of serum miR-183 was associated with poor OS in EOC patients, and proved to be a potential biomarker for EOC. In EOC cell lines, functional assays demonstrated that miR-183 downregulation inhibited cancer proliferation, enhanced bufalin sensitivity and reduced tumorigenicity in vivo. Conclusion MiR-183 may be a prognostic biomarker for EOC, and inhibiting miR-183 may have therapeutic effect to inhibit tumor growth in EOC. PMID:27186298

  20. 3D multiplexed immunoplasmonics microscopy

    NASA Astrophysics Data System (ADS)

    Bergeron, Éric; Patskovsky, Sergiy; Rioux, David; Meunier, Michel

    2016-07-01

    Selective labelling, identification and spatial distribution of cell surface biomarkers can provide important clinical information, such as distinction between healthy and diseased cells, evolution of a disease and selection of the optimal patient-specific treatment. Immunofluorescence is the gold standard for efficient detection of biomarkers expressed by cells. However, antibodies (Abs) conjugated to fluorescent dyes remain limited by their photobleaching, high sensitivity to the environment, low light intensity, and wide absorption and emission spectra. Immunoplasmonics is a novel microscopy method based on the visualization of Abs-functionalized plasmonic nanoparticles (fNPs) targeting cell surface biomarkers. Tunable fNPs should provide higher multiplexing capacity than immunofluorescence since NPs are photostable over time, strongly scatter light at their plasmon peak wavelengths and can be easily functionalized. In this article, we experimentally demonstrate accurate multiplexed detection based on the immunoplasmonics approach. First, we achieve the selective labelling of three targeted cell surface biomarkers (cluster of differentiation 44 (CD44), epidermal growth factor receptor (EGFR) and voltage-gated K+ channel subunit KV1.1) on human cancer CD44+ EGFR+ KV1.1+ MDA-MB-231 cells and reference CD44- EGFR- KV1.1+ 661W cells. The labelling efficiency with three stable specific immunoplasmonics labels (functionalized silver nanospheres (CD44-AgNSs), gold (Au) NSs (EGFR-AuNSs) and Au nanorods (KV1.1-AuNRs)) detected by reflected light microscopy (RLM) is similar to the one with immunofluorescence. Second, we introduce an improved method for 3D localization and spectral identification of fNPs based on fast z-scanning by RLM with three spectral filters corresponding to the plasmon peak wavelengths of the immunoplasmonics labels in the cellular environment (500 nm for 80 nm AgNSs, 580 nm for 100 nm AuNSs and 700 nm for 40 nm × 92 nm AuNRs). Third, the developed

  1. Detecting miRNA by producing RNA: a sensitive assay that combines rolling-circle DNA polymerization and rolling circle transcription.

    PubMed

    Li, Xuemei; Zheng, Fuwei; Ren, Rui

    2015-08-01

    Target miRNA was detected by producing RNA: rolling circle polymerization (RCP) and rolling circle transcription (RCT) were interlinked to provide dual amplification, producing multiplied malachite green (MG) aptamers, and a signal was generated by the SERS (surface-enhanced Raman scattering) quantification of the MG molecules that were bound to the transcripts. PMID:26120604

  2. Development and validation of a multiplex reverse transcription quantitative PCR (RT-qPCR) assay for the rapid detection of Citrus tristeza virus, Citrus psorosis virus, and Citrus leaf blotch virus.

    PubMed

    Osman, Fatima; Hodzic, Emir; Kwon, Sun-Jung; Wang, Jinbo; Vidalakis, Georgios

    2015-08-01

    A single real-time multiplex reverse transcription quantitative polymerase chain reaction (RT-qPCR) assay for the simultaneous detection of Citrus tristeza virus (CTV), Citrus psorosis virus (CPsV), and Citrus leaf blotch virus (CLBV) was developed and validated using three different fluorescently labeled minor groove binding qPCR probes. To increase the detection reliability, coat protein (CP) genes from large number of different isolates of CTV, CPsV and CLBV were sequenced and a multiple sequence alignment was generated with corresponding CP sequences from the GenBank and a robust multiplex RT-qPCR assay was designed. The capacity of the multiplex RT-qPCR assay in detecting the viruses was compared to singleplex RT-qPCR designed specifically for each virus and was assessed using multiple virus isolates from diverse geographical regions and citrus species as well as graft-inoculated citrus plants infected with various combination of the three viruses. No significant difference in detection limits was found and specificity was not affected by the inclusion of the three assays in a multiplex RT-qPCR reaction. Comparison of the viral load for each virus using singleplex and multiplex RT-qPCR assays, revealed no significant differences between the two assays in virus detection. No significant difference in Cq values was detected when using one-step and two-step multiplex RT-qPCR detection formats. Optimizing the RNA extraction technique for citrus tissues and testing the quality of the extracted RNA using RT-qPCR targeting the cytochrome oxidase citrus gene as an RNA specific internal control proved to generate better diagnostic assays. Results showed that the developed multiplex RT-qPCR can streamline viruses testing of citrus nursery stock by replacing three separate singleplex assays, thus reducing time and labor while retaining the same sensitivity and specificity. The three targeted RNA viruses are regulated pathogens for California's mandatory "Section 3701

  3. Development and validation of a multiplex reverse transcription quantitative PCR (RT-qPCR) assay for the rapid detection of Citrus tristeza virus, Citrus psorosis virus, and Citrus leaf blotch virus.

    PubMed

    Osman, Fatima; Hodzic, Emir; Kwon, Sun-Jung; Wang, Jinbo; Vidalakis, Georgios

    2015-08-01

    A single real-time multiplex reverse transcription quantitative polymerase chain reaction (RT-qPCR) assay for the simultaneous detection of Citrus tristeza virus (CTV), Citrus psorosis virus (CPsV), and Citrus leaf blotch virus (CLBV) was developed and validated using three different fluorescently labeled minor groove binding qPCR probes. To increase the detection reliability, coat protein (CP) genes from large number of different isolates of CTV, CPsV and CLBV were sequenced and a multiple sequence alignment was generated with corresponding CP sequences from the GenBank and a robust multiplex RT-qPCR assay was designed. The capacity of the multiplex RT-qPCR assay in detecting the viruses was compared to singleplex RT-qPCR designed specifically for each virus and was assessed using multiple virus isolates from diverse geographical regions and citrus species as well as graft-inoculated citrus plants infected with various combination of the three viruses. No significant difference in detection limits was found and specificity was not affected by the inclusion of the three assays in a multiplex RT-qPCR reaction. Comparison of the viral load for each virus using singleplex and multiplex RT-qPCR assays, revealed no significant differences between the two assays in virus detection. No significant difference in Cq values was detected when using one-step and two-step multiplex RT-qPCR detection formats. Optimizing the RNA extraction technique for citrus tissues and testing the quality of the extracted RNA using RT-qPCR targeting the cytochrome oxidase citrus gene as an RNA specific internal control proved to generate better diagnostic assays. Results showed that the developed multiplex RT-qPCR can streamline viruses testing of citrus nursery stock by replacing three separate singleplex assays, thus reducing time and labor while retaining the same sensitivity and specificity. The three targeted RNA viruses are regulated pathogens for California's mandatory "Section 3701

  4. Dynamic optically multiplexed imaging

    NASA Astrophysics Data System (ADS)

    Rachlin, Yaron; Shah, Vinay; Shepard, R. Hamilton; Shih, Tina

    2015-09-01

    Optically multiplexed imagers overcome the tradeoff between field of view and resolution by superimposing images from multiple fields of view onto a single focal plane. In this paper, we consider the implications of independently shifting each field of view at a rate exceeding the frame rate of the focal plane array and with a precision that can exceed the pixel pitch. A sequence of shifts enables the reconstruction of the underlying scene, with the number of frames required growing inversely with the number of multiplexed images. As a result, measurements from a sufficiently fast sampling sensor can be processed to yield a low distortion image with more pixels than the original focal plane array, a wider field of view than the original optical design, and an aspect ratio different than the original lens. This technique can also enable the collection of low-distortion, wide field of view videos. A sequence of sub-pixel spatial shifts extends this capability to allow the recovery of a wide field of view scene at sub-pixel resolution. To realize this sensor concept, a novel and compact divided aperture multiplexed sensor, capable of rapidly and precisely shifting its fields of view, was prototyped. Using this sensor, we recover twenty-four megapixel images from a four-megapixel focal plane and show the feasibility of simultaneous de-multiplexing and super-resolution.

  5. Label-Free and Enzyme-Free Homogeneous Electrochemical Biosensing Strategy Based on Hybridization Chain Reaction: A Facile, Sensitive, and Highly Specific MicroRNA Assay.

    PubMed

    Hou, Ting; Li, Wei; Liu, Xiaojuan; Li, Feng

    2015-11-17

    Homogenous electrochemical biosensing strategies have attracted substantial attention, because of their advantages of being immobilization-free and having rapid response and improved recognition efficiency, compared to heterogeneous biosensors; however, the high cost of labeling and the strict reaction conditions of tool enzymes associated with current homogeneous electrochemical methods limit their potential applications. To address these issues, herein we reported, for the first time, a simple label-free and enzyme-free homogeneous electrochemical strategy based on hybridization chain reaction (HCR) for sensitive and highly specific detection of microRNA (miRNA). The target miRNA triggers the HCR of two species of metastable DNA hairpin probes, resulting in the formation of multiple G-quadruplex-incorporated long duplex DNA chains. Thus, with the electrochemical indicator Methylene Blue (MB) selectively intercalated into the duplex DNA chain and the multiple G-quadruplexes, a significant electrochemical signal drop is observed, which is dependent on the concentration of the target miRNA. Thus, using this "signal-off" mode, a simple, label-free and enzyme-free homogeneous electrochemical strategy for sensitive miRNA assay is readily realized. This strategy also exhibits excellent selectivity to distinguish even single-base mismatched miRNA. Furthermore, this method also exhibits additional advantages of simplicity and low cost, since both expensive labeling and sophisticated probe immobilization processes are avoided. Therefore, the as-proposed label-free and enzyme-free homogeneous electrochemical strategy may become an alternative method for simple, sensitive, and selective miRNA detection, and it has great potential to be applied in miRNA-related clinical diagnostics and biochemical research.

  6. Combining Single RNA Sensitive Probes with Subdiffraction-Limited and Live-Cell Imaging Enables the Characterization of Virus Dynamics in Cells

    PubMed Central

    2013-01-01

    The creation of fluorescently labeled viruses is currently limited by the length of imaging observation time (e.g., labeling an envelope protein) and the rescue of viral infectivity (e.g., encoding a GFP protein). Using single molecule sensitive RNA hybridization probes delivered to the cytoplasm of infected cells, we were able to isolate individual, infectious, fluorescently labeled human respiratory syncytial virus virions. This was achieved without affecting viral mRNA expression, viral protein expression, or infectivity. Measurements included the characterization of viral proteins and genomic RNA in a single virion using dSTORM, the development of a GFP fusion assay, and the development of a pulse-chase assay for viral RNA production that allowed for the detection of both initial viral RNA and nascent RNA production at designated times postinfection. Live-cell measurements included imaging and characterization of filamentous virion fusion and the quantification of virus replication within the same cell over an eight-hour period. Using probe-labeled viruses, individual viral particles can be characterized at subdiffraction-limited resolution, and viral infections can be quantified in single cells over an entire cycle of replication. The implication of this development is that MTRIP labeling of viral RNA during virus assembly has the potential to become a general methodology for the labeling and study of many important RNA viruses. PMID:24351207

  7. Development of novel AllGlo-probe-based one-step multiplex qRT-PCR assay for rapid identification of avian influenza virus H7N9.

    PubMed

    Zhang, Yanjun; Mao, Haiyan; Yan, Juying; Wang, Xinying; Zhang, Lei; Guus, Koch; Li, Hui; Li, Zhen; Chen, Yin; Gong, Liming; Chen, Zhiping; Xia, Shichang

    2014-07-01

    Recently, human deaths have resulted from infection with low-pathogenicity avian influenza virus H7N9 strains that have emerged recently in China. To strengthen H7N9 surveillance and outbreak control, rapid and reliable diagnostic methods are needed. To develop a sensitive quantitative real-time RT-PCR assay for rapid detection of H7N9 viral RNA, primers and AllGlo probes were designed to target the HA and NA genes of H7N9. Conserved sequences in the HA and NA genes were identified by phylogenic analysis and used as targets for H7N9 virus detection. The similarities of the targeted HA and NA gene sequences from different H7 and N9 influenza virus strains were 93.2-99.9 % and 96.0-99.6 %, respectively The specificity and sensitivity of the new multiplex real-time qRT-PCR was established. The test was used for the detection of viral RNA in human pharyngeal swabs and environmental samples. The detection limit of the multiplex qRT-PCR was estimated to be about 10(-1) TCID50/reaction. Finally, the diagnostic sensitivities of the multiplex qRT-PCR, virus isolation and TaqMan qRT-PCR were compared using pharyngeal swabs and environmental samples. These analyses yielded positive results in 46.7 %, 43.3 % and 20.0 % of the samples, respectively. The novel multiplex AllGlo qRT-PCR is a rapid and sensitive method to identify H7N9 virus in clinical and environmental samples and can be used to facilitate studies on the epidemiology of H7N9 virus.

  8. Extracting information from multiplex networks.

    PubMed

    Iacovacci, Jacopo; Bianconi, Ginestra

    2016-06-01

    Multiplex networks are generalized network structures that are able to describe networks in which the same set of nodes are connected by links that have different connotations. Multiplex networks are ubiquitous since they describe social, financial, engineering, and biological networks as well. Extending our ability to analyze complex networks to multiplex network structures increases greatly the level of information that is possible to extract from big data. For these reasons, characterizing the centrality of nodes in multiplex networks and finding new ways to solve challenging inference problems defined on multiplex networks are fundamental questions of network science. In this paper, we discuss the relevance of the Multiplex PageRank algorithm for measuring the centrality of nodes in multilayer networks and we characterize the utility of the recently introduced indicator function Θ̃(S) for describing their mesoscale organization and community structure. As working examples for studying these measures, we consider three multiplex network datasets coming for social science. PMID:27368796

  9. Extracting information from multiplex networks.

    PubMed

    Iacovacci, Jacopo; Bianconi, Ginestra

    2016-06-01

    Multiplex networks are generalized network structures that are able to describe networks in which the same set of nodes are connected by links that have different connotations. Multiplex networks are ubiquitous since they describe social, financial, engineering, and biological networks as well. Extending our ability to analyze complex networks to multiplex network structures increases greatly the level of information that is possible to extract from big data. For these reasons, characterizing the centrality of nodes in multiplex networks and finding new ways to solve challenging inference problems defined on multiplex networks are fundamental questions of network science. In this paper, we discuss the relevance of the Multiplex PageRank algorithm for measuring the centrality of nodes in multilayer networks and we characterize the utility of the recently introduced indicator function Θ̃(S) for describing their mesoscale organization and community structure. As working examples for studying these measures, we consider three multiplex network datasets coming for social science.

  10. Extracting information from multiplex networks

    NASA Astrophysics Data System (ADS)

    Iacovacci, Jacopo; Bianconi, Ginestra

    2016-06-01

    Multiplex networks are generalized network structures that are able to describe networks in which the same set of nodes are connected by links that have different connotations. Multiplex networks are ubiquitous since they describe social, financial, engineering, and biological networks as well. Extending our ability to analyze complex networks to multiplex network structures increases greatly the level of information that is possible to extract from big data. For these reasons, characterizing the centrality of nodes in multiplex networks and finding new ways to solve challenging inference problems defined on multiplex networks are fundamental questions of network science. In this paper, we discuss the relevance of the Multiplex PageRank algorithm for measuring the centrality of nodes in multilayer networks and we characterize the utility of the recently introduced indicator function Θ ˜ S for describing their mesoscale organization and community structure. As working examples for studying these measures, we consider three multiplex network datasets coming for social science.

  11. Genome-Wide Analysis of Protein and mRNA Copy Numbers in Single Escherichia coli Cells with Single-Molecule Sensitivity.

    PubMed

    Taniguchi, Yuichi

    2015-01-01

    Single-cell proteomic and transcriptomic analysis is an emerging approach for providing quantitative and comprehensive characterization of gene functions in individual cells. This analysis, however, is often hampered by insufficient sensitivity for detecting low copy gene expression products such as transcription factors and regulators. Here I describe a method for the quantitative genome-wide analysis of single-cell protein and mRNA copy numbers with single molecule sensitivity for the model organism Escherichia coli.

  12. Optimized Ultrasound Conditions for Enhanced Sensitivity of Molecular Beacons in the Detection of MDR1 mRNA in Living Cells.

    PubMed

    Zhou, Qiumei; Ma, Yi; Wang, Zhaohui; Wang, Ke; Liu, Ruonan; Han, Zhihao; Zhang, Min; Li, Siwen; Gu, Yueqing

    2016-03-01

    P-glycoprotein (P-gp), aprognostic indicator for chemotherapy failure, is encoded by multidrug resistance gene (MDR1). MDR1 mRNA expression could serve as a guidance for personalized medicine. However, the traditional PCR process for mRNA measurement is complicated and cannot realize the real-time detection of mRNA in living single cells. In this work, optimized gold nanoparticle-based molecular beacons were employed to determine MDR1 mRNA levels in living cancer cells. To improve detection sensitivity, ultrasound (US) irradiation was applied to facilitate and enhance cellular uptake of hairpin DNA-coated gold nanoparticle (hDAuNP). The US conditions including irradiation power, exposure time, duty cycle, and incubation time were optimized. The slight difference in MDR1 expression manipulated by siRNA silence could be recognized by US assisted hDAuNP beacons; a 10-fold increase of detection sensitivity was achieved compared with the nonultrasound assistance. Meanwhile, the detection cycle could be shortened from 12 to 2 h. Furthermore, this hDAuNP beacon can serve as an antisense agent to down-regulate P-gp expression and to reverse drug resistance of MCF-7/Adr cells to doxorubicin. Our results demonstrated that the MDR1 hDAuNP beacon assisted by US irradiation had great potential to predict chemotherapy sensitivity and to overcome multidrug resistance in cancer cells and was thus a promising tool for individualized medicine.

  13. Optimized Ultrasound Conditions for Enhanced Sensitivity of Molecular Beacons in the Detection of MDR1 mRNA in Living Cells.

    PubMed

    Zhou, Qiumei; Ma, Yi; Wang, Zhaohui; Wang, Ke; Liu, Ruonan; Han, Zhihao; Zhang, Min; Li, Siwen; Gu, Yueqing

    2016-03-01

    P-glycoprotein (P-gp), aprognostic indicator for chemotherapy failure, is encoded by multidrug resistance gene (MDR1). MDR1 mRNA expression could serve as a guidance for personalized medicine. However, the traditional PCR process for mRNA measurement is complicated and cannot realize the real-time detection of mRNA in living single cells. In this work, optimized gold nanoparticle-based molecular beacons were employed to determine MDR1 mRNA levels in living cancer cells. To improve detection sensitivity, ultrasound (US) irradiation was applied to facilitate and enhance cellular uptake of hairpin DNA-coated gold nanoparticle (hDAuNP). The US conditions including irradiation power, exposure time, duty cycle, and incubation time were optimized. The slight difference in MDR1 expression manipulated by siRNA silence could be recognized by US assisted hDAuNP beacons; a 10-fold increase of detection sensitivity was achieved compared with the nonultrasound assistance. Meanwhile, the detection cycle could be shortened from 12 to 2 h. Furthermore, this hDAuNP beacon can serve as an antisense agent to down-regulate P-gp expression and to reverse drug resistance of MCF-7/Adr cells to doxorubicin. Our results demonstrated that the MDR1 hDAuNP beacon assisted by US irradiation had great potential to predict chemotherapy sensitivity and to overcome multidrug resistance in cancer cells and was thus a promising tool for individualized medicine. PMID:26821347

  14. Transcriptional activities of the chloroplast-nuclei and proplastid-nuclei isolated from tobacco exhibit different sensitivities to tagetitoxin: implication of the presence of distinct RNA polymerases.

    PubMed

    Sakai, A; Saito, C; Inada, N; Kuroiwa, T

    1998-09-01

    We examined the effects of tagetitoxin, a potent inhibitor of RNA polymerases from chloroplasts and Escherichia coli, on the transcriptional activities of chloroplast- and proplastid-nuclei (nucleoids) isolated from mature tobacco (Nicotiana tabacum L.) leaves and cultured tobacco cells (line BY-2), respectively. Transcription by the isolated chloroplast-nuclei was effectively inhibited by tagetitoxin (95-99% reduction at 10 microM tagetitoxin), but transcription by the isolated proplastid-nuclei was only partially inhibited (40-50% reduction) by this compound. Southern hybridization experiments revealed that the transcription of various plastid genes (psbA, atpA, rpoB, psaA/B, atpB, rbcL, petB, rpl16, and rrn23) was sensitive to tagetitoxin in the isolated chloroplast-nuclei, whereas the transcription of the same genes was relatively resistant to this compound in the isolated proplastid-nuclei. These results suggest that; (i) distinct RNA polymerase activities with different sensitivities to tagetitoxin are present in plastids, (ii) a tagetitoxin-sensitive RNA polymerase is the major RNA polymerase in chloroplasts whereas a tagetitoxin-insensitive enzyme is major in proplastids, and (iii) both RNA polymerases can transcribe various plastid genes.

  15. Co-delivery of hydrophobic paclitaxel and hydrophilic AURKA specific siRNA by redox-sensitive micelles for effective treatment of breast cancer.

    PubMed

    Yin, Tingjie; Wang, Lei; Yin, Lifang; Zhou, Jianping; Huo, Meirong

    2015-08-01

    In this study, a novel redox-sensitive micellar system constructed from a hyaluronic acid-based amphiphilic conjugate (HA-ss-(OA-g-bPEI), HSOP) was successfully developed for tumor-targeted co-delivery of paclitaxel (PTX) and AURKA specific siRNA (si-AURKA). HSOP exhibited excellent loading capacities for both PTX and siRNA with adjustable dosing ratios and desirable redox-sensitivity independently verified by morphological changes of micelles alongside in vitro release of both drugs in different reducing environments. Moreover, flow cytometry and confocal microscopy analysis confirmed that HSOP micelles were capable of simultaneously delivering PTX and siRNA into MDA-MB-231 breast cancer cells via HA-receptor mediated endocytosis followed by rapid transport of cargoes into the cytosol. Successful delivery and transport amplified the synergistic effects between the drugs while leading to substantially greater antitumor efficacy when compared with single drug-loaded micelles and non-sensitive co-loaded micelles. In vivo investigation demonstrated that HSOP micelles could effectively accumulate in tumor sites and possessed the greatest antitumor efficacy over non-sensitive co-delivery control and redox-sensitive single-drug controls. These findings indicated that redox-sensitive HSOP co-delivery system holds great promise for combined drug/gene treatment for targeted cancer therapy.

  16. mRNA profiling for body fluid identification by reverse transcription endpoint PCR and realtime PCR.

    PubMed

    Haas, C; Klesser, B; Maake, C; Bär, W; Kratzer, A

    2009-03-01

    mRNA profiling is a promising new method for the identification of body fluids from biological stains. Major advantages of mRNA profiling are the possibility of detecting several body fluids in one multiplex reaction and of simultaneously isolating DNA without loss of material. A reverse transcription endpoint polymerase chain reaction (PCR) method and a realtime PCR assay were established for the identification of blood, saliva, semen, vaginal secretions and menstrual blood, and were compared to conventional enzymatic and immunologic tests. The results for specificity, sensitivity and suitability to biological stains were satisfying and mRNA stability was demonstrated for up to 2-year-old stains. Two novel multiplex assays were created with the endpoint PCR primers: multiplex 1 amplifies two markers for each of the above mentioned body fluids and is suited for screening; multiplex 2 was designed for the detection of blood, vaginal secretions and menstrual blood. The results demonstrate that both endpoint PCR and realtime PCR are suitable for the identification of body fluids in forensic stains and represent an effective alternative to conventional enzymatic and immunologic tests.

  17. MicroRNA-101 regulates T-cell acute lymphoblastic leukemia progression and chemotherapeutic sensitivity by targeting Notch1

    PubMed Central

    Qian, Lu; Zhang, Wanggang; Lei, Bo; He, Aili; Ye, Lianhong; Li, Xingzhou; Dong, Xin

    2016-01-01

    The present study aimed to investigate the role of microRNA (miR)-101 in acute lymphoblastic leukemia progression and chemoresistance. Furthermore, a novel target gene of miR-101 was identified. Here, we confirmed that miR-101 was significantly downregulated in the blood samples of patients with T-cell acute lymphoblastic leukemia (T-ALL) compared with the healthy controls, as determined by reverse transcription quantitative polymerase chain reaction (RTqPCR) analysis. The in vitro experiments demonstrated that miR-101 significantly repressed the proliferation and invasion, and induced potent apoptosis in Jurkat cells, as determined by CCK-8, flow cytometer and cell invasion assays. Luciferase assay confirmed that Notch1 was a target gene of miR-101, and western blotting showed that miR-101 suppressed the expression of Notch1 at the protein level. Moreover, functional restoration assays revealed that Notch1 mediates the effects of miR-101 on Jurkat cell proliferation, apoptosis and invasion. miR-101 enhanced the sensitivity of Jurkat cells to the chemotherapeutic agent adriamycin. Taken together, our results show for the first time that miR-101 acts as a tumor suppressor in T-cell acute lymphoblastic leukaemia and it could enhance chemotherapeutic sensitivity. Furthermore, Notch1 was identified to be a novel target of miR-101. This study indicates that miR-101 may represent a potential therapeutic target for T-cell acute lymphoblastic leukemia intervention. PMID:27666896

  18. Effect of salt and water intake on epithelial sodium channel mRNA abundance in the kidney of salt-sensitive Sabra rats.

    PubMed

    Nicco, Carole; Bankir, Lise; Bouby, Nadine

    2003-12-01

    1. The level of mRNA expression of epithelial sodium channel (ENaC) subunits was studied in a salt-dependent hypertensive rat strain (Sabra). These rats exhibit high vasopressin levels compared with their normotensive counterparts. We also investigated whether this expression is influenced by changes in the sodium intake/aldosterone axis or in the fluid intake/vasopressin axis. 2. A higher expression of beta- and gamma-subunit mRNA was found in salt-sensitive compared with salt-resistant rats on a normal salt diet. A high-sodium diet did not alter mRNA abundance in either substrain. In contrast, water supplementation in salt-sensitive rats fed the high-sodium diet induced a marked reduction in mRNA abundance of beta- and gamma-subunits. 3. The present study provides evidence that beta- and gamma-subunits of ENaC are differently expressed in the kidney of salt-sensitive and salt-resistant Sabra rats and that their abundance is regulated by vasopressin, not by sodium intake. These results are consistent with the hypothesis that increased vasopressin-dependent ENaC expression and activity may contribute to the pathogenesis of hypertension in salt-sensitive Sabra rats. PMID:14678237

  19. Temperature-sensitive mutations for live-attenuated Rift Valley fever vaccines: implications from other RNA viruses

    PubMed Central

    Nishiyama, Shoko; Ikegami, Tetsuro

    2015-01-01

    Rift Valley fever (RVF) is a mosquito-borne zoonotic disease endemic to the African continent. RVF is characterized by high rate of abortions in ruminants and hemorrhagic fever, encephalitis, or blindness in humans. RVF is caused by the Rift Valley fever virus (RVFV: genus Phlebovirus, family Bunyaviridae). Vaccination is the only known effective strategy to prevent the disease, but there are no licensed RVF vaccines available for humans. A live-attenuated vaccine candidate derived from the wild-type pathogenic Egyptian ZH548 strain, MP-12, has been conditionally licensed for veterinary use in the U.S. MP-12 displays a temperature-sensitive (ts) phenotype and does not replicate at 41°C. The ts mutation limits viral replication at a specific body temperature and may lead to an attenuation of the virus. Here we will review well-characterized ts mutations for RNA viruses, and further discuss the potential in designing novel live-attenuated vaccines for RVF. PMID:26322023

  20. Screening a yeast library of temperature-sensitive mutants reveals a role for actin in tombusvirus RNA recombination.

    PubMed

    Prasanth, K Reddisiva; Kovalev, Nikolay; de Castro Martín, Isabel Fernández; Baker, Jannine; Nagy, Peter D

    2016-02-01

    Genetic recombination in RNA viruses drives the evolutionary arms race with host's antiviral strategies and recombination also facilitates adaptation of viruses to new hosts. In this paper, the authors used tombusvirus and a temperature-sensitive (ts) mutant library of yeast to identify 40 host proteins affecting viral recombination in yeast model host. Subsequent detailed analysis with two identified actin-related proteins, Act1p and Arp3p, has revealed that the wt actin network helps TBSV to maintain low level viral recombination. Pharmacological inhibition of actin in plant protoplasts confirmed the role of the actin network in tombusvirus recombination. An in vitro approach revealed the altered activity of the tombusvirus replicase in the presence of mutated Act1p. The authors show more efficient recruitment of a cellular DEAD-box helicase, which enhances tombusvirus recombination, into the membrane-bound replicase in Act1p mutant yeast. Overall, this work shows that the actin network affects tombusvirus recombination in yeast and plant cells.

  1. A novel nested multiplex polymerase chain reaction (PCR) assay for differential detection of Entamoeba histolytica, E. moshkovskii and E. dispar DNA in stool samples

    PubMed Central

    Khairnar, Krishna; Parija, Subhash C

    2007-01-01

    Background E. histolytica, a pathogenic amoeba, is indistinguishable in its cyst and trophozoite stages from those of non-pathogenic E. moshkovskii and E. dispar by light microscopy. We have developed a nested multiplex PCR targeting a 16S-like rRNA gene for differential detection of all the three morphologically similar forms of E. histolytica, E. moshkovskii and E. dispar simultaneously in stool samples. Results The species specific product size for E. histolytica, E. moshkovskii and E. dispar was 439, 553 and 174 bp respectively, which was clearly different for all the three Entamoeba species. The nested multiplex PCR showed a sensitivity of 94% and specificity of 100% for the demonstration of E. histolytica, E. moshkovskii and E. dispar DNA in stool samples. The PCR was positive for E. histolytica, E. moshkovskii and E. dispar in a total of 190 out of 202 stool specimens (94% sensitive) that were positive for E. histolytica/E. dispar/E. moshkovskii by examination of stool by microscopy and/or culture. All the 35 negative control stool samples that were negative for E. histolytica/E. dispar/E. moshkovskii by microscopy and culture were also found negative by the nested multiplex PCR (100% specific). The result from the study shows that only 34.6% of the patient stool samples that were positive for E. histolytica/E. dispar/E. moshkovskii by examination of stool by microscopy and/or culture, were actually positive for pathogenic E. histolytica and the remaining majority of the stool samples were positive for non-pathogenic E. dispar or E. moshkovskii as demonstrated by the use of nested multiplex PCR. Conclusion The present study reports a new nested multiplex PCR strategy for species specific detection and differentiation of E. histolytica, E. dispar and E. moshkovskii DNA in stool specimens. The test is highly specific, sensitive and also rapid, providing the results within 12 hours of receiving stool specimens. PMID:17524135

  2. Sensitive localized surface plasmon resonance multiplexing protocols.

    PubMed

    Jia, Kun; Bijeon, Jean L; Adam, Pierre M; Ionescu, Rodica E

    2012-09-18

    Herein are reported two new protocols to obtain different zones of localized surface plasmon resonance (LSPR) gold nanostructures on single glass substrate by using a vacuum evaporation technique followed by a high-temperature annealing (550 °C). The thickness of the gold film, considered as the essential parameter to determine specific LSPR properties, is successfully modulated. In the first protocol, a metal mask is integrated onto the glass substrate during vacuum evaporation to vary the gold film thickness by a "shadowing effect", while in the second protocol several evaporation cycles (up to four cycles) at predefined areas onto the single substrate are performed. The resulting gold-modified samples are characterized using a transmission UV-vis extinction optical setup and scanning electron microscopy (SEM). The size distribution histograms of nanoparticles are also acquired. By employing the first protocol, thanks to the presence of different zones of gold nanoparticles on a single substrate, optimized LSPR responses to different (bio)functionalization zones are rapidly screened. Independently, the second protocol exhibited an excellent correlation between the nominative evaporated gold film thickness, gold nanoparticle sizes, and plasmonic properties (resonant wavelength and peak amplitude). Such substrates are further used in the construction of LSPR immunosensors for the detection of atrazine herbicide.

  3. Highly selective and sensitive detection of miRNA based on toehold-mediated strand displacement reaction and DNA tetrahedron substrate.

    PubMed

    Li, Wei; Jiang, Wei; Ding, Yongshun; Wang, Lei

    2015-09-15

    MicroRNAs (miRNAs) play important roles in a variety of biological processes and have been regarded as tumor biomarkers in cancer diagnosis and prognosis. In this work, a single-molecule counting method for miRNA analysis was proposed based on toehold-mediated strand displacement reaction (SDR) and DNA tetrahedron substrate. Firstly, a specially designed DNA tetrahedron was assembled with a hairpin at one of the vertex, which has an overhanging toehold domain. Then, the DNA tetrahedron was immobilized on the epoxy-functional glass slide by epoxy-amine reaction, forming a DNA tetrahedron substrate. Next, the target miRNA perhybridized with the toehold domain and initiated a strand displacement reaction along with the unfolding of the hairpin, realizing the selective recognization of miRNA. Finally, a biotin labeled detection DNA was hybridized with the new emerging single strand and the streptavidin coated QDs were used as fluorescent probes. Fluorescent images were acquired via epi-fluorescence microscopy, the numbers of fluorescence dots were counted one by one for quantification. The detection limit is 5 fM, which displayed an excellent sensitivity. Moreover, the proposed method which can accurately be identified the target miRNA among its family members, demonstrated an admirable selectivity. Furthermore, miRNA analysis in total RNA samples from human lung tissues was performed, suggesting the feasibility of this method for quantitative detection of miRNA in biomedical research and early clinical diagnostics.

  4. Developmental changes in the hypothalamic mRNA levels of prepro-orexin and orexin receptors and their sensitivity to fasting in male and female rats.

    PubMed

    Iwasa, Takeshi; Matsuzaki, Toshiya; Munkhzaya, Munkhsaikhan; Tungalagsuvd, Altankhuu; Kuwahara, Akira; Yasui, Toshiyuki; Irahara, Minoru

    2015-11-01

    Orexin, which is also called as hypocretin (Hcrt), a product of the prepro-orexin (pp-orexin//Hcrt) gene, affects various physiological and behavioral functions, such as the sleep-wake cycle and appetite. The developmental changes in the hypothalamic mRNA levels of pp-prexin and the orexin receptors OX1R and OX2R and their sensitivity to fasting were evaluated in both male and female rats. During development, hypothalamic pp-orexin/Hcrt mRNA expression increased in both male and female rats, whereas hypothalamic OX1R mRNA expression decreased in both sexes. In addition, hypothalamic OX2R mRNA expression increased in male rats, but did not change in female rats. Fasting did not affect hypothalamic pp-orexin/Hcrt mRNA expression in either sex. Hypothalamic OX1R mRNA expression was increased by fasting in the prepubertal period (postnatal days 20 and 30) in female rats, but was not affected by fasting in males. In male rats, hypothalamic OX2R mRNA expression was decreased by fasting during the neonatal period (postnatal day 10), but not the prepubertal period (postnatal days 20 and 30). In females, hypothalamic OX2R mRNA expression was also decreased by fasting; however, the fasting-induced downregulation of hypothalamic OX2R expression persisted until postnatal day 20. These results indicate that the developmental patterns of components of the orexin system and their sensitivity to fasting during the neonatal and prepubertal periods only differ slightly between the sexes. These differences might be involved in the development of some physiological and behavioral functions.

  5. Multiplex detection of mutations.

    PubMed

    Perlin, David S; Balashov, Sergey; Park, Steven

    2008-01-01

    Rapid and reliable detection of mutations at the genetic level is an integral part of modern molecular diagnostics. These mutations can range from dominant single nucleotide polymorphisms within specific loci to codominant heterozygotic insertions and they present considerable challenges to investigators in developing rapid nucleic acid-based amplification assays that can distinguish wild-type from mutant alleles. The recent improvements of real-time polymerase chain reaction (PCR) using self-reporting fluorescence probes have given researchers a powerful tool in developing assays for mutation detection that can be multiplexed for high-throughput screening of multiple mutations and cost effectiveness. Here we describe an application of a multiplexed real-time PCR assay using Molecular Beacon probes for the detection of mutations in codon 54 of the CYP51A gene in Aspergillus fumigatus conferring triazole resistance.

  6. Multiplex data bus simulator

    SciTech Connect

    Garbo, D.L.

    1983-01-01

    A multiplex data-bus simulator for analyzing multiprocessor designs is presented. The simulator was designed to be user-friendly, thus allowing a multiprocessor designer to enter various configuration inputs in a concise and orderly fashion through the use of menus. The designer is also provided a method of visualizing a message traffic flow through the use of graphical representation of events. 3 references.

  7. On-chip noninterference angular momentum multiplexing of broadband light.

    PubMed

    Ren, Haoran; Li, Xiangping; Zhang, Qiming; Gu, Min

    2016-05-13

    Angular momentum division has emerged as a physically orthogonal multiplexing method in high-capacity optical information technologies. However, the typical bulky elements used for information retrieval from the overall diffracted field, based on the interference method, impose a fundamental limit toward realizing on-chip multiplexing. We demonstrate noninterference angular momentum multiplexing by using a mode-sorting nanoring aperture with a chip-scale footprint as small as 4.2 micrometers by 4.2 micrometers, where nanoring slits exhibit a distinctive outcoupling efficiency on tightly confined plasmonic modes. The nonresonant mode-sorting sensitivity and scalability of our approach enable on-chip parallel multiplexing over a bandwidth of 150 nanometers in the visible wavelength range. The results offer the possibility of ultrahigh-capacity and miniaturized nanophotonic devices harnessing angular momentum division.

  8. Burst noise in the HAWAII-1RG multiplexer

    NASA Astrophysics Data System (ADS)

    Bacon, Candice M.; McMurtry, Craig W.; Pipher, Judith L.; Forrest, William J.; Garnett, James D.

    2005-08-01

    Burst noise (also known as popcorn noise and random telegraph signal/noise) is a phenomenon that is understood to be a result of defects in the vicinity of a p-n junction. It is characterized by rapid level shifts in both positive and negative directions and can have varying magnitudes. This noise has been seen in both HAWAII-1RG and HAWAII-2RG multiplexers and is under investigation. We have done extensive burst noise testing on a HAWAII-1RG multiplexer, where we have determined a significant percentage of pixels exhibit the phenomenon. In addition, the prevalence of small magnitude transitions make sensitivity of detection the main limiting factor. Since this is a noise source for the HAWAII-1RG multiplexer, its elimination would make the HAWAII-1RG and the HAWAII-2RG even lower noise multiplexers.

  9. On-chip noninterference angular momentum multiplexing of broadband light.

    PubMed

    Ren, Haoran; Li, Xiangping; Zhang, Qiming; Gu, Min

    2016-05-13

    Angular momentum division has emerged as a physically orthogonal multiplexing method in high-capacity optical information technologies. However, the typical bulky elements used for information retrieval from the overall diffracted field, based on the interference method, impose a fundamental limit toward realizing on-chip multiplexing. We demonstrate noninterference angular momentum multiplexing by using a mode-sorting nanoring aperture with a chip-scale footprint as small as 4.2 micrometers by 4.2 micrometers, where nanoring slits exhibit a distinctive outcoupling efficiency on tightly confined plasmonic modes. The nonresonant mode-sorting sensitivity and scalability of our approach enable on-chip parallel multiplexing over a bandwidth of 150 nanometers in the visible wavelength range. The results offer the possibility of ultrahigh-capacity and miniaturized nanophotonic devices harnessing angular momentum division. PMID:27056843

  10. On-chip noninterference angular momentum multiplexing of broadband light

    NASA Astrophysics Data System (ADS)

    Ren, Haoran; Li, Xiangping; Zhang, Qiming; Gu, Min

    2016-05-01

    Angular momentum division has emerged as a physically orthogonal multiplexing method in high-capacity optical information technologies. However, the typical bulky elements used for information retrieval from the overall diffracted field, based on the interference method, impose a fundamental limit toward realizing on-chip multiplexing. We demonstrate noninterference angular momentum multiplexing by using a mode-sorting nanoring aperture with a chip-scale footprint as small as 4.2 micrometers by 4.2 micrometers, where nanoring slits exhibit a distinctive outcoupling efficiency on tightly confined plasmonic modes. The nonresonant mode-sorting sensitivity and scalability of our approach enable on-chip parallel multiplexing over a bandwidth of 150 nanometers in the visible wavelength range. The results offer the possibility of ultrahigh-capacity and miniaturized nanophotonic devices harnessing angular momentum division.

  11. 3D multiplexed immunoplasmonics microscopy.

    PubMed

    Bergeron, Éric; Patskovsky, Sergiy; Rioux, David; Meunier, Michel

    2016-07-21

    Selective labelling, identification and spatial distribution of cell surface biomarkers can provide important clinical information, such as distinction between healthy and diseased cells, evolution of a disease and selection of the optimal patient-specific treatment. Immunofluorescence is the gold standard for efficient detection of biomarkers expressed by cells. However, antibodies (Abs) conjugated to fluorescent dyes remain limited by their photobleaching, high sensitivity to the environment, low light intensity, and wide absorption and emission spectra. Immunoplasmonics is a novel microscopy method based on the visualization of Abs-functionalized plasmonic nanoparticles (fNPs) targeting cell surface biomarkers. Tunable fNPs should provide higher multiplexing capacity than immunofluorescence since NPs are photostable over time, strongly scatter light at their plasmon peak wavelengths and can be easily functionalized. In this article, we experimentally demonstrate accurate multiplexed detection based on the immunoplasmonics approach. First, we achieve the selective labelling of three targeted cell surface biomarkers (cluster of differentiation 44 (CD44), epidermal growth factor receptor (EGFR) and voltage-gated K(+) channel subunit KV1.1) on human cancer CD44(+) EGFR(+) KV1.1(+) MDA-MB-231 cells and reference CD44(-) EGFR(-) KV1.1(+) 661W cells. The labelling efficiency with three stable specific immunoplasmonics labels (functionalized silver nanospheres (CD44-AgNSs), gold (Au) NSs (EGFR-AuNSs) and Au nanorods (KV1.1-AuNRs)) detected by reflected light microscopy (RLM) is similar to the one with immunofluorescence. Second, we introduce an improved method for 3D localization and spectral identification of fNPs based on fast z-scanning by RLM with three spectral filters corresponding to the plasmon peak wavelengths of the immunoplasmonics labels in the cellular environment (500 nm for 80 nm AgNSs, 580 nm for 100 nm AuNSs and 700 nm for 40 nm × 92 nm AuNRs). Third

  12. Simultaneous multiplex PCR detection of seven cucurbit-infecting viruses.

    PubMed

    Kwon, Ji Yeon; Hong, Jin Sung; Kim, Min Jea; Choi, Sun Hee; Min, Byeong Eun; Song, Eun Gyeong; Kim, Hyun Hee; Ryu, Ki Hyun

    2014-09-01

    Two multiplex polymerase chain reaction (PCR) systems using dual priming oligonucleotide (DPO) primers were developed for the simultaneous detection of seven cucurbit-infecting viruses. One system allows for the detection of papaya ringspot virus, watermelon mosaic virus, and zucchini yellow mosaic virus, whereas the other permits the detection of cucumber green mottle mosaic virus, cucumber fruit mottle mosaic virus, kyuri green mottle mosaic virus, and zucchini green mottle mosaic virus. Viral species-specific DPO primers developed in this study detected as little as 10 fg/μl of viral RNA under monoplex conditions and 10 pg/μl of viral RNA under multiplex conditions. Multiplex PCR using the DPO primer sets was capable of amplifying viral genes at annealing temperatures ranging from 53 °C to 63 °C. Whereas the use of conventional primers gave rise to non-specific bands, the DPO primers detected target viral genes in the absence of non-specific amplification. When these DPO multiplex primer sets were applied to virus-infected cucurbit samples obtained in the field, multiple infection as well as single infection was accurately identified. This novel approach could also detect multiple viruses in infected seeds. The reliability of multiplex PCR systems using DPO primers for plant virus detection is discussed.

  13. Simultaneous multiplex PCR detection of seven cucurbit-infecting viruses.

    PubMed

    Kwon, Ji Yeon; Hong, Jin Sung; Kim, Min Jea; Choi, Sun Hee; Min, Byeong Eun; Song, Eun Gyeong; Kim, Hyun Hee; Ryu, Ki Hyun

    2014-09-01

    Two multiplex polymerase chain reaction (PCR) systems using dual priming oligonucleotide (DPO) primers were developed for the simultaneous detection of seven cucurbit-infecting viruses. One system allows for the detection of papaya ringspot virus, watermelon mosaic virus, and zucchini yellow mosaic virus, whereas the other permits the detection of cucumber green mottle mosaic virus, cucumber fruit mottle mosaic virus, kyuri green mottle mosaic virus, and zucchini green mottle mosaic virus. Viral species-specific DPO primers developed in this study detected as little as 10 fg/μl of viral RNA under monoplex conditions and 10 pg/μl of viral RNA under multiplex conditions. Multiplex PCR using the DPO primer sets was capable of amplifying viral genes at annealing temperatures ranging from 53 °C to 63 °C. Whereas the use of conventional primers gave rise to non-specific bands, the DPO primers detected target viral genes in the absence of non-specific amplification. When these DPO multiplex primer sets were applied to virus-infected cucurbit samples obtained in the field, multiple infection as well as single infection was accurately identified. This novel approach could also detect multiple viruses in infected seeds. The reliability of multiplex PCR systems using DPO primers for plant virus detection is discussed. PMID:24937806

  14. Self-calibrating multiplexer circuit

    DOEpatents

    Wahl, Chris P.

    1997-01-01

    A time domain multiplexer system with automatic determination of acceptable multiplexer output limits, error determination, or correction is comprised of a time domain multiplexer, a computer, a constant current source capable of at least three distinct current levels, and two series resistances employed for calibration and testing. A two point linear calibration curve defining acceptable multiplexer voltage limits may be defined by the computer by determining the voltage output of the multiplexer to very accurately known input signals developed from predetermined current levels across the series resistances. Drift in the multiplexer may be detected by the computer when the output voltage limits, expected during normal operation, are exceeded, or the relationship defined by the calibration curve is invalidated.

  15. Anti-EphA10 antibody-conjugated pH-sensitive liposomes for specific intracellular delivery of siRNA.

    PubMed

    Zang, Xinlong; Ding, Huaiwei; Zhao, Xiufeng; Li, Xiaowei; Du, Zhouqi; Hu, Haiyang; Qiao, Mingxi; Chen, Dawei; Deng, Yuihui; Zhao, Xiuli

    2016-01-01

    Therapeutic delivery of small interfering RNA (siRNA) is a major challenge that limits its potential clinical application. Here, a pH-sensitive cholesterol-Schiff base-polyethylene glycol (Chol-SIB-PEG)-modified cationic liposome-siRNA complex, conjugated with the recombinant humanized anti-EphA10 antibody (Eph), was developed as an efficient nonviral siRNA delivery system. Chol-SIB-PEG was successfully synthesized and confirmed with FTIR and (1)H-NMR. An Eph-PEG-SIB-Chol-modified liposome-siRNA complex (EPSLR) was prepared and characterized by size, zeta potential, gel retardation, and encapsulation efficiency. Electrophoresis results showed that EPSLR was resistant to heparin replacement and protected siRNA from fetal bovine serum digestion. EPSLR exhibited only minor cytotoxicity in MCF-7/ADR cells. The results of flow cytometry and confocal laser scanning microscopy suggested that EPSLR enhanced siRNA transfection in MCF-7/ADR cells. Intracellular distribution experiment revealed that EPSLR could escape from the endo-lysosomal organelle and release siRNA into cytoplasm at 4 hours posttransfection. Western blot experiment demonstrated that EPSLR was able to significantly reduce the levels of MDR1 protein in MCF-7/ADR cells. The in vivo study of DIR-labeled complexes in mice bearing MCF-7/ADR tumor indicated that EPSLR could reach the tumor site rather than other organs more effectively. All these results demonstrate that EPSLR has much potential for effective siRNA delivery and may facilitate its therapeutic application.

  16. Anti-EphA10 antibody-conjugated pH-sensitive liposomes for specific intracellular delivery of siRNA.

    PubMed

    Zang, Xinlong; Ding, Huaiwei; Zhao, Xiufeng; Li, Xiaowei; Du, Zhouqi; Hu, Haiyang; Qiao, Mingxi; Chen, Dawei; Deng, Yuihui; Zhao, Xiuli

    2016-01-01

    Therapeutic delivery of small interfering RNA (siRNA) is a major challenge that limits its potential clinical application. Here, a pH-sensitive cholesterol-Schiff base-polyethylene glycol (Chol-SIB-PEG)-modified cationic liposome-siRNA complex, conjugated with the recombinant humanized anti-EphA10 antibody (Eph), was developed as an efficient nonviral siRNA delivery system. Chol-SIB-PEG was successfully synthesized and confirmed with FTIR and (1)H-NMR. An Eph-PEG-SIB-Chol-modified liposome-siRNA complex (EPSLR) was prepared and characterized by size, zeta potential, gel retardation, and encapsulation efficiency. Electrophoresis results showed that EPSLR was resistant to heparin replacement and protected siRNA from fetal bovine serum digestion. EPSLR exhibited only minor cytotoxicity in MCF-7/ADR cells. The results of flow cytometry and confocal laser scanning microscopy suggested that EPSLR enhanced siRNA transfection in MCF-7/ADR cells. Intracellular distribution experiment revealed that EPSLR could escape from the endo-lysosomal organelle and release siRNA into cytoplasm at 4 hours posttransfection. Western blot experiment demonstrated that EPSLR was able to significantly reduce the levels of MDR1 protein in MCF-7/ADR cells. The in vivo study of DIR-labeled complexes in mice bearing MCF-7/ADR tumor indicated that EPSLR could reach the tumor site rather than other organs more effectively. All these results demonstrate that EPSLR has much potential for effective siRNA delivery and may facilitate its therapeutic application. PMID:27574425

  17. Anti-EphA10 antibody-conjugated pH-sensitive liposomes for specific intracellular delivery of siRNA

    PubMed Central

    Zang, Xinlong; Ding, Huaiwei; Zhao, Xiufeng; Li, Xiaowei; Du, Zhouqi; Hu, Haiyang; Qiao, Mingxi; Chen, Dawei; Deng, Yuihui; Zhao, Xiuli

    2016-01-01

    Therapeutic delivery of small interfering RNA (siRNA) is a major challenge that limits its potential clinical application. Here, a pH-sensitive cholesterol–Schiff base–polyethylene glycol (Chol–SIB–PEG)-modified cationic liposome–siRNA complex, conjugated with the recombinant humanized anti-EphA10 antibody (Eph), was developed as an efficient nonviral siRNA delivery system. Chol–SIB–PEG was successfully synthesized and confirmed with FTIR and 1H-NMR. An Eph–PEG–SIB–Chol-modified liposome–siRNA complex (EPSLR) was prepared and characterized by size, zeta potential, gel retardation, and encapsulation efficiency. Electrophoresis results showed that EPSLR was resistant to heparin replacement and protected siRNA from fetal bovine serum digestion. EPSLR exhibited only minor cytotoxicity in MCF-7/ADR cells. The results of flow cytometry and confocal laser scanning microscopy suggested that EPSLR enhanced siRNA transfection in MCF-7/ADR cells. Intracellular distribution experiment revealed that EPSLR could escape from the endo-lysosomal organelle and release siRNA into cytoplasm at 4 hours posttransfection. Western blot experiment demonstrated that EPSLR was able to significantly reduce the levels of MDR1 protein in MCF-7/ADR cells. The in vivo study of DIR-labeled complexes in mice bearing MCF-7/ADR tumor indicated that EPSLR could reach the tumor site rather than other organs more effectively. All these results demonstrate that EPSLR has much potential for effective siRNA delivery and may facilitate its therapeutic application. PMID:27574425

  18. Influenza virus-specific RNA and protein syntheses in cells infected with temperature-sensitive mutants defective in the genome segment encoding nonstructural proteins.

    PubMed

    Wolstenholme, A J; Barrett, T; Nichol, S T; Mahy, B W

    1980-07-01

    Virus-specific protein and RNA syntheses have been analyzed in chicken embryo fibroblast cells infected with two group IV temperature-sensitive (ts) mutants of influenza A (fowl plague) virus in which the ts lesion maps in RNA segment 8 (J. W. Almond, D. McGeoch, and R. D. Barry, Virology 92:416-427, 1979), known to code to code for two nonstructural proteins, NS1 and NS2. Both mutants induced the synthesis of similar amounts of all the early virus-specific proteins (P1, P2, P3, NP, and NS1) at temperatures that were either permissive (34 degrees C) or nonpermissive (40.5 degrees C) for replication. However, the synthesis of M protein, which normally accumulates late in infection, was greatly reduced in ts mutant-infected cells at 40.5 degrees C compared to 34 degrees C. The NS2 protein was not detected at either temperature in cells infected with one mutant (mN3), and was detected only at the permissive temperature in cells infected with mutant ts47. There was no overall reduction in polyadenylated (A+) complementary RNA, which functions as mRNA, in cells infected with these mutants at 40.5 degrees C compared to 34 degrees C, nor was there any evidence of selective accumulation of this type of RNA within the nucleus at the nonpermissive temperature. No significant differences in ts mutant virion RNA transcriptase activity were detected by assays in vitro at 31 and 40.5 degrees C compared to wild-type virus. Virus-specific non-polyadenylated (A-) complementary RNA, which is believed to act as the template for new virion RNA production, accumulated normally in cells at both 34 and 40.5 degrees C, but at 40.5 degrees C accumulation of new virion RNA was reduced by greater than 90% when compared to accumulation at 34 degrees C.

  19. Multiplex assays to diagnose celiac disease.

    PubMed

    Lochman, Ivo; Martis, Peter; Burlingame, Rufus W; Lochmanová, Alexandra

    2007-08-01

    Patients with celiac disease are sensitive to the gluten fractions of wheat. Symptoms include gastrointestinal problems and a failure to thrive in children, but may range from headaches to general malaise in adults. Thus, it is difficult to diagnose celiac disease by symptoms alone. The standard diagnostic criteria include the presence of the characteristic anti-gliadin or anti-tissue transglutaminase antibodies (anti-tTG) in serum, flattened mucosa on intestinal biopsy, and improved symptoms on a gluten-free diet. Because of the ease of use of the tTG enzyme-linked immunosorbent assay (ELISA) compared to endomysial by indirect immunofluorescence assay, there has been much more screening for celiac disease in recent years. This increased screening showed that celiac disease was more prevalent than previously believed. We compared a new multiplex assay that includes a novel form of deamidated gliadin and recombinant human tTG as the antigens to other assays using standard antigens. In addition, the new assay detects the presence of selective IgA deficiency, which shows a 10-fold increase in prevalence in patients with celiac disease compared to the general population. The combination of sensitivity and specificity of the new multiplex assay was equal or better than those for standard assays. Thus the performance, ease of use, and ability to measure three clinically important parameters in a single test make the new multiplex assay a viable alternative to standard assays in a clinical lab.

  20. A rapid and sensitive non-radioactive method applicable for genome-wide analysis of Saccharomyces cerevisiae genes involved in small RNA biology.

    PubMed

    Wu, Jingyan; Huang, Hsiao-Yun; Hopper, Anita K

    2013-04-01

    Conventional isolation and detection methods for small RNAs from yeast cells have been designed for a limited number of samples. In order to be able to conduct a genome-wide assessment of how each gene product impacts upon small RNAs, we developed a rapid method for analysing small RNAs from Saccharomyces cerevisiae wild-type (wt) and mutants cells in the deletion and temperature-sensitive (ts) collections. Our method implements three optimized techniques: a procedure for growing small yeast cultures in 96-deepwell plates, a fast procedure for small RNA isolation from the plates, and a sensitive non-radioactive northern method for RNA detection. The RNA isolation procedure requires only 4 h for processing 96 samples, is highly reproducible and yields RNA of good quality and quantity. The non-radioactive northern method employs digoxigenin (DIG)-labelled DNA probes and chemiluminescence. It detects femtomole levels of small RNAs within 1 min exposure time. We minimized the processing time for large-scale analysis and optimized the stripping and reprobing procedures for analyses of multiple RNAs from a single membrane. The method described is rapid, sensitive, safe and cost-effective for genome-wide screens of novel genes involved in the biogenesis, subcellular trafficking and stability of small RNAs. Moreover, it will be useful to educational laboratory class venues and to research institutions with limited access to radioisotopes or robots.

  1. Chopped molecular beam multiplexing system

    NASA Technical Reports Server (NTRS)

    Adams, Billy R. (Inventor)

    1986-01-01

    The integration of a chopped molecular beam mass spectrometer with a time multiplexing system is described. The chopping of the molecular beam is synchronized with the time intervals by a phase detector and a synchronous motor. Arithmetic means are generated for phase shifting the chopper with respect to the multiplexer. A four channel amplifier provides the capacity to independently vary the baseline and amplitude in each channel of the multiplexing system.

  2. Abnormal expression of ENaC and SGK1 mRNA induced by dietary sodium in Dahl salt-sensitively hypertensive rats.

    PubMed

    Aoi, Wataru; Niisato, Naomi; Sawabe, Yukinori; Miyazaki, Hiroaki; Tokuda, Shinsaku; Nishio, Kyosuke; Yoshikawa, Toshikazu; Marunaka, Yoshinori

    2007-10-01

    Epithelial sodium channel (ENaC) plays a crucial role in controlling sodium reabsorption in the kidney keeping the normal blood pressure. We previously reported that the expression of ENaC mRNA in the kidney of Dahl salt-sensitive (DS) rats was abnormally regulated by aldosterone, however it is unknown if dietary sodium affects the expression of ENaC and serum and glucocorticoid-regulated kinase 1 (SGK1), which plays an important role in ENaC activation, in DS rats. In the present study, we investigated whether dietary sodium abnormally affects the expression of ENaC and SGK1 mRNA in DS rats. DS and Dahl salt-resistant (DR) rats (8 weeks old) were divided into three different groups, respectively: (1) low sodium diet (0.005% NaCl), (2) normal sodium diet (0.3% NaCl), and (3) high sodium diet (8% NaCl). The high sodium diet for 4 weeks in DS rats elevated the systolic blood pressure, but did not in any other groups. The expression of alpha-ENaC mRNA in DS rats was abnormally increased by high sodium diet in contrast to DR rats, while it was normally increased by low sodium diet in DS rats similar to DR rats. The expression of beta- and gamma-ENaC mRNA in DS rats was also abnormally increased by high sodium diet unlike DR rats. The expression of SGK1 mRNA was elevated by high sodium diet in DS rats, but it was decreased in DR rats. These observations indicate that the expression of ENaC and SGK1 mRNA is abnormally regulated by dietary sodium in salt-sensitively hypertensive rats, and that this abnormal expression would be one of the factors causing salt-sensitive hypertension.

  3. Chimeric adenoviral vector Ad5/F35-mediated APE1 siRNA enhances sensitivity of human colorectal cancer cells to radiotherapy in vitro and in vivo.

    PubMed

    Xiang, D-B; Chen, Z-T; Wang, D; Li, M-X; Xie, J-Y; Zhang, Y-S; Qing, Y; Li, Z-P; Xie, J

    2008-10-01

    Apurinic/apyrimidinic endonuclease (APE1), a bifunctional AP endonuclease/redox factor, is important in DNA repair and redox signaling, may be associated with radioresistance. Here we investigate whether targeted inhibition of APE1 can sensitize tumor cells to irradiation in vitro and in vivo. We first constructed chimeric adenoviral vector Ad5/F35 carrying human APE1 siRNA (Ad5/F35-APE1 siRNA). The infectivity of chimeric Ad5/F35 to LOVO colon cancer cells was greater than that of Ad5. APE1 was strongly expressed and nuclear factor kappaB (NF-kappaB), a downstream molecule of APE1, known as a radioresistance factor, was constitutively active in LOVO cells. Infection of LOVO cells with Ad5/F35-APE1 siRNA resulted in a dose-dependent decrease of APE1 protein and AP endonuclease activity in vitro. Ad5/F35-APE1 siRNA significantly enhanced sensitivity of LOVO cells to irradiation in clonogenic survival assays, associated with increased cell apoptosis. The APE1 expression in LOVO cells was induced by irradiation in a dose-dependent manner, accompanied with the enhancement of DNA-binding activity of NF-kappaB and Ad5/F35-APE1 siRNA effectively inhibited constitutive and irradiation-induced APE1 expression and NF-kappaB activation. In a subcutaneous nude mouse colon cancer model, Ad5/F35-APE1 siRNA (5 x 10(8) IU, intratumoral injection) inhibited the expression of APE1 protein in LOVO xenografts, and significantly enhanced inhibition of tumor growth by irradiation. In conclusion, APE1 may be involved as one of the radioresistance factors, and targeted inhibition of APE1 shows an effective means of enhancing tumor sensitivity to radiotherapy.

  4. Dual phase multiplex polymerase chain reaction

    DOEpatents

    Pemov, Alexander; Bavykin, Sergei

    2008-10-07

    Highly specific and sensitive methods were developed for multiplex amplification of nucleic acids on supports such as microarrays. Based on a specific primer design, methods include five types of amplification that proceed in a reaction chamber simultaneously. These relate to four types of multiplex amplification of a target DNA on a solid support, directed by forward and reverse complex primers immobilized to the support and a fifth type--pseudo-monoplex polymerase chain reaction (PCR) of multiple targets in solution, directed by a single pair of unbound universal primers. The addition of the universal primers in the reaction mixture increases the yield over the traditional "bridge" amplification on a solid support by approximately ten times. Methods that provide multitarget amplification and detection of as little as 0.45-4.5.times.10.sup.-12 g (equivalent to 10.sup.2-10.sup.3 genomes) of a bacterial genomic DNA are disclosed.

  5. Sensitive detection of microRNA in complex biological samples via enzymatic signal amplification using DNA polymerase coupled with nicking endonuclease.

    PubMed

    Yin, Bin-Cheng; Liu, Yu-Qiang; Ye, Bang-Ce

    2013-12-01

    MicroRNA (miRNA) has become an ideal biomarker candidate for cancer diagnosis, prognosis, and therapy. In this study, we have developed a novel one-step method for sensitive and specific miRNA detection via enzymatic signal amplification and demonstrated its practical application in biological samples. The proposed signal amplification strategy is an integrated "biological circuit" designed to initiate a cascade of enzymatic polymerization reactions in order to detect, amplify, and measure a specific miRNA sequence by using the isothermal strand-displacement property of a mesophilic DNA polymerase together with the nicking activity of a restriction endonuclease. The circuit is composed of two molecular switches operating in series: the nicking endonuclease-assisted isothermal polymerization reaction activated by a specific miRNA and the strand-displacement polymerization reaction designed to initiate molecular beacon-assisted amplification and signal transduction. The hsa-miR-141 (miR-141) was chosen as a target miRNA because its level specifically elevates in prostate cancer. The proposed method allowed quantitative sequence-specific detection of miR-141 in a dynamic range from 1 fM to 100 nM, with an excellent ability to discriminate differences among miR-200 family members. Moreover, the detection assay was applied to quantify miR-141 in cancerous cell lysates. The results are in excellent agreement with those from the reverse transcription polymerase chain reaction method. On the basis of these findings, we believe that this proposed sensitive and specific assay has great potential as a miRNA quantification method for use in biomedical research and clinical diagnosis.

  6. MicroRNA-144 affects radiotherapy sensitivity by promoting proliferation, migration and invasion of breast cancer cells.

    PubMed

    Yu, Lei; Yang, Yanming; Hou, Jiguang; Zhai, Chengwei; Song, Yunhao; Zhang, Zhiliang; Qiu, Ling; Jia, Xiaojing

    2015-10-01

    Radiotherapy resistance remains a major obstacle for patients with breast cancer. miRNAs are important regulators in many biological processes including proliferation, apoptosis, invasion and metastasis and response to treatment in different types of tumors. Here, we describe the role of miRNA-144 in the regulation of radiotherapy sensitivity, migration and invasion of breast cancer cells. The cell survival rate of breast cancer cells was measured by WST-1 assay after irradiation. The caspase-3/-7 activity and apoptotic proteins were analyzed by Caspase-Glo3/7 assay and western blot analysis, respectively. The migration and invasion of breast cancer cells were evaluated by BD Transwell migration and Matrigel invasion assays. The EMT markers were detected by western blot analysis. We found that overexpression of miR-144 increased the proliferation rate of MDA-MB-231 cells without radiation. Both MDA-MB‑231 and SKBR3 cells exhibited significantly increased radiation resistance after overexpression of miR-144. Meanwhile, the migration and invasion of both MDA-MB-231 and SKBR3 cells were changed by altered miR-144 expression. In addition, the overexpression of miR-144 inhibited E-cadherin expression and promoted Snail expression. miR-144 activated AKT by downregulation of PTEN in breast cancer cells. Our results strongly suggest that miR-144 acts as an important regulator of tumorigenesis and tumor progression of breast cancer. These results indicate that miR-144 might serve as a potential molecular target for breast cancer treatment.

  7. Evaluation of Nanogen MGB Alert Detection Reagents in a multiplex real-time PCR for influenza virus types A and B and respiratory syncytial virus.

    PubMed

    Hymas, Weston C; Hillyard, David R

    2009-03-01

    A multiplex real-time RT-PCR assay that detects influenza A, influenza B and respiratory syncytial virus (RSV) using the MGB Alert Influenza A&B/RSV Detection Reagent RUO (Nanogen, San Diego, CA) was developed. The Nanogen detection reagents consist of PCR primers and minor groove binder-conjugated hybridization probes for each virus and an internal control. Virus typing was determined by post-PCR melt curve analysis. A non-competitive armored RNA internal control was co-extracted with each sample to monitor nucleic acid extraction and RT-PCR. The assay was evaluated using a collection of culture, DFA and RT-PCR (Hexaplex, Prodesse, Waukesha, WI) positive and negative samples. The real-time multiplex assay detected 236 of 237 positive specimens for a 99% correlation. Of 30 Hexaplex negative samples tested, the multiplex real-time assay detected an additional 7 positives confirmed using additional PCR assays. Melt curve analysis for each virus produced average melting peaks of 60.4 degrees C, 66.7 degrees C and 69.4 degrees C for influenza A, influenza B and RSV respectively. Sequence analysis of 7 influenza A samples producing aberrant melt curves, confirmed the presence of a single nucleotide polymorphism beneath the influenza A probe. The limit of detection of each virus in 4 different sample types was measured to be between 7 and 806 copies. Overall, the multiplexed real-time RT-PCR assay was sensitive, robust and easy to use.

  8. Development of a multiplex assay to measure the effects of shipping and storage conditions on the quality of RNA used in molecular assays for detection of viral haemorrhagic septicemia virus.

    PubMed

    Siah, A; Duesund, H; Frisch, K; Nylund, A; McKenzie, P; Saksida, S

    2014-09-01

    Abstract In routine diagnostics, real-time reverse transcriptase quantitative PCR (RT-qPCR) has become a powerful method for fish health screening. Collection, transportation, and storage conditions of specimens could dramatically affect their integrity and could consequently affect RT-qPCR test results. In this study, to assess the expression profile of elongation factor 1 alpha (ELF-1α) gene, head kidney (HK) tissues from Atlantic Salmon Salmo salar were exposed at room temperature, 4°C, -20°C, and -80°C as well as in 70% ethanol for 6, 12, 24, 48, and 72 h. Data showed a significant increase of RT-qPCR cycle threshold (Ct) values for ELF-1α ranging from 14.7 to 26.5 cycles for tissues exposed to room temperature. In order to mimic the sample transportation conditions, different temperatures of storage were used and tissue quality was evaluated using ELF-1α gene expression. Data showed that Ct values for ELF-1α increased significantly when the tissues were transported on ice for 2 h, stored at -20°C, thawed on ice for 6 h, and stored again at -80°C. The HK tissues collected from Atlantic Salmon challenged with viral hemorrhagic septicemia virus (VHSV) through intraperitoneal injection were exposed at room temperature for 0, 6, 12, 24, 48, 72, and 96 h. Data showed a good correlation of values for ELF-1α and VHSV Ct although the ELF-1α mRNA of the host degraded faster than the RNA of VHSV. Based on these data, HK tissues could be transported on ice or ice packs without the quality of the tissue being affected when stored at -80°C upon arrival at the laboratory. In addition, 70% ethanol could be used as a preservative for long-distance transportation. For an efficient diagnostic test, a duplex VHSV-ELF-1α was developed and optimized. Data showed that the sensitivity of the duplex assay for VHSV was similar to the singleplex. Received November 25, 2013; accepted February 14, 2014. PMID:25229489

  9. 3D multiplexed immunoplasmonics microscopy

    NASA Astrophysics Data System (ADS)

    Bergeron, Éric; Patskovsky, Sergiy; Rioux, David; Meunier, Michel

    2016-07-01

    Selective labelling, identification and spatial distribution of cell surface biomarkers can provide important clinical information, such as distinction between healthy and diseased cells, evolution of a disease and selection of the optimal patient-specific treatment. Immunofluorescence is the gold standard for efficient detection of biomarkers expressed by cells. However, antibodies (Abs) conjugated to fluorescent dyes remain limited by their photobleaching, high sensitivity to the environment, low light intensity, and wide absorption and emission spectra. Immunoplasmonics is a novel microscopy method based on the visualization of Abs-functionalized plasmonic nanoparticles (fNPs) targeting cell surface biomarkers. Tunable fNPs should provide higher multiplexing capacity than immunofluorescence since NPs are photostable over time, strongly scatter light at their plasmon peak wavelengths and can be easily functionalized. In this article, we experimentally demonstrate accurate multiplexed detection based on the immunoplasmonics approach. First, we achieve the selective labelling of three targeted cell surface biomarkers (cluster of differentiation 44 (CD44), epidermal growth factor receptor (EGFR) and voltage-gated K+ channel subunit KV1.1) on human cancer CD44+ EGFR+ KV1.1+ MDA-MB-231 cells and reference CD44- EGFR- KV1.1+ 661W cells. The labelling efficiency with three stable specific immunoplasmonics labels (functionalized silver nanospheres (CD44-AgNSs), gold (Au) NSs (EGFR-AuNSs) and Au nanorods (KV1.1-AuNRs)) detected by reflected light microscopy (RLM) is similar to the one with immunofluorescence. Second, we introduce an improved method for 3D localization and spectral identification of fNPs based on fast z-scanning by RLM with three spectral filters corresponding to the plasmon peak wavelengths of the immunoplasmonics labels in the cellular environment (500 nm for 80 nm AgNSs, 580 nm for 100 nm AuNSs and 700 nm for 40 nm × 92 nm AuNRs). Third, the developed

  10. Highly sensitive and selective microRNA detection based on DNA-bio-bar-code and enzyme-assisted strand cycle exponential signal amplification.

    PubMed

    Dong, Haifeng; Meng, Xiangdan; Dai, Wenhao; Cao, Yu; Lu, Huiting; Zhou, Shufeng; Zhang, Xueji

    2015-04-21

    Herein, a highly sensitive and selective microRNA (miRNA) detection strategy using DNA-bio-bar-code amplification (BCA) and Nb·BbvCI nicking enzyme-assisted strand cycle for exponential signal amplification was designed. The DNA-BCA system contains a locked nucleic acid (LNA) modified DNA probe for improving hybridization efficiency, while a signal reported molecular beacon (MB) with an endonuclease recognition site was designed for strand cycle amplification. In the presence of target miRNA, the oligonucleotides functionalized magnetic nanoprobe (MNP-DNA) and gold nanoprobe (AuNP-DNA) with numerous reported probes (RP) can hybridize with target miRNA, respectively, to form a sandwich structure. After sandwich structures were separated from the solution by the magnetic field, the RP were released under high temperature to recognize the MB and cleaved the hairpin DNA to induce the dissociation of RP. The dissociated RP then triggered the next strand cycle to produce exponential fluorescent signal amplification for miRNA detection. Under optimized conditions, the exponential signal amplification system shows a good linear range of 6 orders of magnitude (from 0.3 pM to 3 aM) with limit of detection (LOD) down to 52.5 zM, while the sandwich structure renders the system with high selectivity. Meanwhile, the feasibility of the proposed strategy for cell miRNA detection was confirmed by analyzing miRNA-21 in HeLa lysates. Given the high-performance for miRNA analysis, the strategy has a promising application in biological detection and in clinical diagnosis.

  11. Rapid Multiplex Nested PCR for Detection of Respiratory Viruses▿

    PubMed Central

    Lam, W. Y.; Yeung, Apple C. M.; Tang, Julian W.; Ip, Margaret; Chan, Edward W. C.; Hui, Mamie; Chan, Paul K. S.

    2007-01-01

    Respiratory tract infections can be caused by a heterogeneous group of viruses and bacteria that produce similar clinical presentations. Specific diagnosis therefore relies on laboratory investigation. This study developed and evaluated five groups of multiplex nested PCR assays that could simultaneously detect 21 different respiratory pathogens: influenza A virus (H1N1, H3N2, and H5N1); influenza B virus; parainfluenza virus types 1, 2, 3, 4a, and 4b; respiratory syncytial virus A and B; human rhinoviruses; human enteroviruses; human coronaviruses OC43 and 229E; severe acute respiratory syndrome coronavirus; human metapneumoviruses; Mycoplasma pneumoniae; Chlamydophila pneumoniae; Legionella pneumophila; and adenoviruses (A to F). These multiplex nested PCRs adopted fast PCR technology. The high speed of fast PCR (within 35 min) greatly improved the efficiency of these assays. The results show that these multiplex nested PCR assays are specific and more sensitive (100- to 1,000-fold) than conventional methods. Among the 303 clinical specimens tested, the multiplex nested PCR achieved an overall positive rate of 48.5% (95% confidence interval [CI], 42.9 to 54.1%), which was significantly higher than that of virus isolation (20.1% [95% CI, 15.6 to 24.6%]) and that of direct detection by immunofluorescence assay (13.5% [95% CI, 9.7 to 17.4%]). The improved sensitivity was partly due to the higher sensitivity of multiplex nested PCR than that of conventional methods in detecting cultivatable viruses. Moreover, the ability of the multiplex nested PCR to detect noncultivatable viruses, particularly rhinoviruses, coronavirus OC43, and metapneumoviruses, contributed a major gain (15.6%) in the overall positive rate. In conclusion, rapid multiplex nested PCR assays can improve the diagnostic yield for respiratory infections to allow prompt interventive actions to be taken. PMID:17804659

  12. Detecting respiratory viral RNA using expanded genetic alphabets and self-avoiding DNA.

    PubMed

    Glushakova, Lyudmyla G; Sharma, Nidhi; Hoshika, Shuichi; Bradley, Andrea C; Bradley, Kevin M; Yang, Zunyi; Benner, Steven A

    2015-11-15

    Nucleic acid (NA)-targeted tests detect and quantify viral DNA and RNA (collectively xNA) to support epidemiological surveillance and, in individual patients, to guide therapy. They commonly use polymerase chain reaction (PCR) and reverse transcription PCR. Although these all have rapid turnaround, they are expensive to run. Multiplexing would allow their cost to be spread over multiple targets, but often only with lower sensitivity and accuracy, noise, false positives, and false negatives; these arise by interactions between the multiple nucleic acid primers and probes in a multiplexed kit. Here we offer a multiplexed assay for a panel of respiratory viruses that mitigates these problems by combining several nucleic acid analogs from the emerging field of synthetic biology: (i) self-avoiding molecular recognition systems (SAMRSs), which facilitate multiplexing, and (ii) artificially expanded genetic information systems (AEGISs), which enable low-noise PCR. These are supplemented by "transliteration" technology, which converts standard nucleotides in a target to AEGIS nucleotides in a product, improving hybridization. The combination supports a multiplexed Luminex-based respiratory panel that potentially differentiates influenza viruses A and B, respiratory syncytial virus, severe acute respiratory syndrome coronavirus (SARS), and Middle East respiratory syndrome (MERS) coronavirus, detecting as few as 10 MERS virions in a 20-μl sample. PMID:26299645

  13. Multiplex Flow Assays

    PubMed Central

    2016-01-01

    Lateral flow or dipstick assays (e.g., home pregnancy tests), where an analyte solution is drawn through a porous membrane and is detected by localization onto a capture probe residing at a specific site on the flow strip, are the most commonly and extensively used type of diagnostic assay. However, after over 30 years of use, these assays are constrained to measuring one or a few analytes at a time. Here, we describe a completely general method, in which any single-plex lateral flow assay is transformed into a multiplex assay capable of measuring an arbitrarily large number of analytes simultaneously. Instead of identifying the analyte by its localization onto a specific geometric location in the flow medium, the analyte-specific capture probe is identified by its association with a specific optically encoded region within the flow medium. The capture probes for nucleic acids, antigens, or antibodies are attached to highly porous agarose beads, which have been encoded using multiple lanthanide emitters to create a unique optical signature for each capture probe. The optically encoded capture probe-derivatized beads are placed in contact with the analyte-containing porous flow medium and the analytes are captured onto the encoded regions as the solution flows through the porous medium. To perform a multiplex diagnostic assay, a solution comprising multiple analytes is passed through the flow medium containing the capture probe-derivatized beads, and the captured analyte is treated with a suitable fluorescent reporter. We demonstrate this multiplex analysis technique by simultaneously measuring DNA samples, antigen–antibody pairs, and mixtures of multiple nucleic acids and antibodies.

  14. Sensitivity-Enhanced MQ-HCN-CCH-TOCSY and MQ-HCN-CCH-COSY Pulse Schemes for 13C/ 15N Labeled RNA Oligonucleotides

    NASA Astrophysics Data System (ADS)

    Hu, Weidong; Jiang, Licong; Gosser, Yuying Q.

    2000-07-01

    Sensitivity enhanced multiple-quantum 3D HCN-CCH-TOCSY and HCN-CCH-COSY experiments are presented for the ribose resonance assignment of 13C/15N-labeled RNA sample. The experiments make use of the chemical shift dispersion of N1/N9 of pyrimidine/purine to distinguish the ribose spin systems. They provide a complementary approach for the assignment of ribose resonance to the currently used HCCH-COSY and HCCH-TOCSY type experiments in which either 13C or 1H is utilized to separate the different ribose spin systems. The pulse schemes have been demonstrated on a 23-mer 13C/15N-labeled RNA aptamer complexed with neomycin and tested on a 32-mer RNA complexed with a 23-residue peptide.

  15. Hardware Counter Multiplexing

    2000-10-13

    The Hardware Counter Multiplexer works with the built-in counter registers on computer processors. These counters record various low-level events as software runs, but they can not record all possible events at the same time. This software helps work around that limitation by counting a series of different events in sequence over a period of time. This in turn allows programmers to measure interesting combinations of events, rather than single events. The software is designed tomore » work with multithreaded or single-threaded programs.« less

  16. Multiplexed capillary electrophoresis system

    DOEpatents

    Yeung, E.S.; Li, Q.; Lu, X.

    1998-04-21

    The invention provides a side-entry optical excitation geometry for use in a multiplexed capillary electrophoresis system. A charge-injection device is optically coupled to capillaries in the array such that the interior of a capillary is imaged onto only one pixel. In Sanger-type 4-label DNA sequencing reactions, nucleotide identification (``base calling``) is improved by using two long-pass filters to split fluorescence emission into two emission channels. A binary poly(ethyleneoxide) matrix is used in the electrophoretic separations. 19 figs.

  17. Multiplexed capillary electrophoresis system

    DOEpatents

    Yeung, E.S.; Chang, H.T.; Fung, E.N.; Li, Q.; Lu, X.

    1996-12-10

    The invention provides a side-entry optical excitation geometry for use in a multiplexed capillary electrophoresis system. A charge-injection device is optically coupled to capillaries in the array such that the interior of a capillary is imaged onto only one pixel. In Sanger-type 4-label DNA sequencing reactions, nucleotide identification (``base calling``) is improved by using two long-pass filters to split fluorescence emission into two emission channels. A binary poly(ethyleneoxide) matrix is used in the electrophoretic separations. 19 figs.

  18. Multiplexed capillary electrophoresis system

    DOEpatents

    Yeung, Edward S.; Li, Qingbo; Lu, Xiandan

    1998-04-21

    The invention provides a side-entry optical excitation geometry for use in a multiplexed capillary electrophoresis system. A charge-injection device is optically coupled to capillaries in the array such that the interior of a capillary is imaged onto only one pixel. In Sanger-type 4-label DNA sequencing reactions, nucleotide identification ("base calling") is improved by using two long-pass filters to split fluorescence emission into two emission channels. A binary poly(ethyleneoxide) matrix is used in the electrophoretic separations.

  19. Multiplexed capillary electrophoresis system

    DOEpatents

    Yeung, Edward S.; Chang, Huan-Tsang; Fung, Eliza N.; Li, Qingbo; Lu, Xiandan

    1996-12-10

    The invention provides a side-entry optical excitation geometry for use in a multiplexed capillary electrophoresis system. A charge-injection device is optically coupled to capillaries in the array such that the interior of a capillary is imaged onto only one pixel. In Sanger-type 4-label DNA sequencing reactions, nucleotide identification ("base calling") is improved by using two long-pass filters to split fluorescence emission into two emission channels. A binary poly(ethyleneoxide) matrix is used in the electrophoretic separations.

  20. A multiplex real-time PCR panel assay for simultaneous detection and differentiation of 12 common swine viruses.

    PubMed

    Shi, Xiju; Liu, Xuming; Wang, Qin; Das, Amaresh; Ma, Guiping; Xu, Lu; Sun, Qing; Peddireddi, Lalitha; Jia, Wei; Liu, Yanhua; Anderson, Gary; Bai, Jianfa; Shi, Jishu

    2016-10-01

    Mixed infection with different pathogens is common in swine production systems especially under intensive production conditions. Quick and accurate detection and differentiation of different pathogens are necessary for epidemiological surveillance, disease management and import and export controls. In this study, we developed and validated a panel of multiplex real-time PCR/RT-PCR assays composed of four subpanels, each detects three common swine pathogens. The panel detects 12 viruses or viral serotypes, namely, VSV-IN, VSV-NJ, SVDV, CSFV, ASFV, FMDV, PCV2, PPV, PRV, PRRSV-NA, PRRSV-EU and SIV. Correlation coefficients (R(2)) and PCR amplification efficiencies of all singular and triplex real-time PCR reactions are within the acceptable range. Comparison between singular and triplex real-time PCR assays of each subpanel indicates that there is no significant interference on assay sensitivities caused by multiplexing. Specificity tests on 226 target clinical samples or 4 viral strains and 91 non-target clinical samples revealed that the real-time PCR panel is 100% specific, and there is no cross amplification observed. The limit of detection of each triplex real-time PCR is less than 10 copies per reaction for DNA, and less than 16 copies per reaction for RNA viruses. The newly developed multiplex real-time PCR panel also detected different combinations of co-infections as confirmed by other means of detections.

  1. A multiplex real-time PCR panel assay for simultaneous detection and differentiation of 12 common swine viruses.

    PubMed

    Shi, Xiju; Liu, Xuming; Wang, Qin; Das, Amaresh; Ma, Guiping; Xu, Lu; Sun, Qing; Peddireddi, Lalitha; Jia, Wei; Liu, Yanhua; Anderson, Gary; Bai, Jianfa; Shi, Jishu

    2016-10-01

    Mixed infection with different pathogens is common in swine production systems especially under intensive production conditions. Quick and accurate detection and differentiation of different pathogens are necessary for epidemiological surveillance, disease management and import and export controls. In this study, we developed and validated a panel of multiplex real-time PCR/RT-PCR assays composed of four subpanels, each detects three common swine pathogens. The panel detects 12 viruses or viral serotypes, namely, VSV-IN, VSV-NJ, SVDV, CSFV, ASFV, FMDV, PCV2, PPV, PRV, PRRSV-NA, PRRSV-EU and SIV. Correlation coefficients (R(2)) and PCR amplification efficiencies of all singular and triplex real-time PCR reactions are within the acceptable range. Comparison between singular and triplex real-time PCR assays of each subpanel indicates that there is no significant interference on assay sensitivities caused by multiplexing. Specificity tests on 226 target clinical samples or 4 viral strains and 91 non-target clinical samples revealed that the real-time PCR panel is 100% specific, and there is no cross amplification observed. The limit of detection of each triplex real-time PCR is less than 10 copies per reaction for DNA, and less than 16 copies per reaction for RNA viruses. The newly developed multiplex real-time PCR panel also detected different combinations of co-infections as confirmed by other means of detections. PMID:27506582

  2. Expression of microRNA-30a-5p in drug-resistant and drug-sensitive ovarian cancer cell lines

    PubMed Central

    Liu, Jin; Wu, Xiaohua; Liu, Hongmei; Liang, Yijuan; Gao, Xinping; Cai, Zhihui; Wang, Weiming; Zhang, Hui

    2016-01-01

    The present study aimed to explore the expression of microRNA (miRNA or miR) in drug-resistant and drug-sensitive ovarian cancer cell lines, and to seek the potential therapeutic target of ovarian cancer drug-resistant mechanism in order to improve drug resistance by altering miRNA levels. The drug-resistant characteristics of SKOV3/DDP, SKOV3, COC1/DDP and COC1 cell lines were studied. The miRNAs that were differentially expressed between cisplatin-resistant cells and its parental cells in ovarian cancer were screened with a miRNA chip. The effect of miRNAs was detected, and their drug-resistant mechanism was investigated by transfection and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide methods. Among the expression screening of miRNAs, 41 mRNAs, including Homo sapiens (hsa)-miR-30a-5p and hsa-miR-34c-5p, were highly expressed in the drug-resistant cells, whereas 44 miRNAs, including hsa-miR-96-5p and hsa-miR-200c-3p, were lowly expressed. The expression levels of hsa-miR-30a-5p in two types of ovarian cancer chemotherapy-resistant cell lines were significantly higher than those in chemotherapy-sensitive cell lines, which was associated with ovarian cancer chemotherapy resistance. In conclusion, high expression of miRNA-30a-5p was able to promote cell growth and colony forming ability, and enhance cell migration and invasion. Thus, miRNA-30a-5p is expected to become a meaningful novel target for ovarian cancer resistant treatment. PMID:27602140

  3. Shift-multiplexing complex spectral-domain optical coherence tomography

    NASA Astrophysics Data System (ADS)

    Huang, Haochong; Jiang, Zhuqing; Wang, Dayong; Cai, Wenyuan; Man, Tianlong; Wang, Zhe; Panezai, Spozmai

    2014-01-01

    We propose and experimentally demonstrate a shift-multiplexing complex spectral-domain optical coherence tomography (shift-multiplexing CSD-OCT) method, in which the maximum detection depth of SD-OCT can be greatly extended by incorporating the shift-multiplexing of detection positions with CSD-OCT. The tomographic imaging with twofold or threefold microscopic slides as the target sample is performed. The experimental results show that the tomographic imaging with more uniform brightness and clarity for the different depth regions in a thick sample can be achieved by the shift-multiplexing CSD-OCT system. In particular, even while the sample's depth is beyond the maximum imaging depth of CSD-OCT system, the tomographic imaging of this sample can still be realized by using the shift-multiplexing CSD-OCT method without the need for any replacement of the equipment, such as high spectral capacity grating or high resolution of CCD. The shift-multiplexing CSD-OCT system can perform the imaging with the optimization and less reduction of sensitivity for the deeper detection position in the sample.

  4. Selective inhibition of FLICE-like inhibitory protein expression with small interfering RNA oligonucleotides is sufficient to sensitize tumor cells for TRAIL-induced apoptosis.

    PubMed Central

    Siegmund, Daniela; Hadwiger, Philipp; Pfizenmaier, Klaus; Vornlocher, Hans-Peter; Wajant, Harald

    2002-01-01

    BACKGROUND: Most tumors express death receptors and their activation represents a potential selective approach in cancer treatment. The most promising candidate for tumor selective death receptor-activation is tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)/Apo2L, which activates the death receptors TRAIL-R1 and TRAIL-R2, and induces apoptosis preferentially in tumor cells but not in normal tissues. However, many cancer cells are not or only moderately sensitive towards TRAIL and require cotreatment with irradiation or chemotherapy to yield a therapeutically reasonable apoptotic response. Because chemotherapy can have a broad range of unwanted side effects, more specific means for sensitizing tumor cells for TRAIL are desirable. The expression of the cellular FLICE-like inhibitory protein (cFLIP) is regarded as a major cause of TRAIL resistance. We therefore analyzed the usefulness of targeting FLIP to sensitize tumor cells for TRAIL-induced apoptosis. MATERIALS AND METHODS: To selectively interfere with expression of cFLIP short double-stranded RNA oligonucleotides (small interfering RNAs [siRNAs]) were introduced in the human cell lines SV80 and KB by electroporation. Effects of siRNA on FLIP expression were analyzed by Western blotting and RNase protection assay and correlated with TRAIL sensitivity upon stimulation with recombinant soluble TRAIL and TRAIL-R1- and TRAIL-R2-specific agonistic antibodies. RESULTS: FLIP expression can be inhibited by RNA interference using siRNAs, evident from reduced levels of FLIP-mRNA and FLIP protein. Inhibition of cFLIP expression sensitizes cells for apoptosis induction by TRAIL and other death ligands. In accordance with the presumed function of FLIP as an inhibitor of death receptor-induced caspase-8 activation, down-regulation of FLIP by siRNAs enhanced TRAIL-induced caspase-8 activation. CONCLUSION: Inhibition of FLIP expression was sufficient to sensitize tumor cells for TRAIL-induced apoptosis. The

  5. The use of pH-sensitive functional selenium nanoparticles shows enhanced in vivo VEGF-siRNA silencing and fluorescence imaging

    NASA Astrophysics Data System (ADS)

    Yu, Qianqian; Liu, Yanan; Cao, Chengwen; Le, Fangling; Qin, Xiuying; Sun, Dongdong; Liu, Jie

    2014-07-01

    The utility of small interfering RNAs (siRNAs) has shown great promise in treating a variety of diseases including many types of cancer. While their ability to silence a wide range of target genes underlies their effectiveness, the application of therapies remains hindered by a lack of an effective delivery system. In this study, we sought to develop an siRNA-delivery system for VEGF, a known signaling molecule involved in cancer, that consists of two selenium nanoparticles SeNPs and G2/PAH-Cit/SeNPs. A G2/PAH-Cit/SeNP is a pH-sensitive delivery system that is capable of enhancing siRNA loading, thus increasing siRNA release efficiency and subsequent target gene silencing both in vitro and in vivo. In vivo experiments using G2/PAH-Cit/SeNPs@siRNA led to significantly higher accumulation of siRNA within the tumor itself, VEGF gene silencing, and reduced angiogenesis in the tumor. Furthermore, the G2/PAH-Cit/SeNP delivery system not only enhanced anti-tumor effects on tumor-bearing nude mice as compared to SeNPs@siRNA, but also resulted in weak occurrence of lesions in major target organs. In sum, this study provides a new class of siRNA delivery system, thereby providing an alternative therapeutic route for cancer treatment.The utility of small interfering RNAs (siRNAs) has shown great promise in treating a variety of diseases including many types of cancer. While their ability to silence a wide range of target genes underlies their effectiveness, the application of therapies remains hindered by a lack of an effective delivery system. In this study, we sought to develop an siRNA-delivery system for VEGF, a known signaling molecule involved in cancer, that consists of two selenium nanoparticles SeNPs and G2/PAH-Cit/SeNPs. A G2/PAH-Cit/SeNP is a pH-sensitive delivery system that is capable of enhancing siRNA loading, thus increasing siRNA release efficiency and subsequent target gene silencing both in vitro and in vivo. In vivo experiments using G2/PAH-Cit/SeNPs@siRNA

  6. Scalable multiplexing for parallel imaging with interleaved optical coherence tomography

    PubMed Central

    Lee, Hee Yoon; Marvdashti, Tahereh; Duan, Lian; Khan, Saara A.; Ellerbee, Audrey K.

    2014-01-01

    We demonstrate highly parallel imaging with interleaved optical coherence tomography (iOCT) using an in-house-fabricated, air-spaced virtually-imaged phased array (VIPA). The air-spaced VIPA performs spectral encoding of the interferograms from multiple lateral points within a single sweep of the source and allows us to tune and balance several imaging parameters: number of multiplexed points, ranging depth, and sensitivity. In addition to a thorough discussion of the parameters and operating principles of the VIPA, we experimentally demonstrate the effect of different VIPA designs on the multiplexing potential of iOCT. Using a 200-kHz light source, we achieve an effective A-scan rate of 3.2-MHz by multiplexing 16 lateral points onto a single wavelength sweep. The improved sensitivity of this system is demonstrated for 3D imaging of biological samples such as a human finger and a fruit fly. PMID:25401031

  7. Rare Earth core/shell nanobarcodes for multiplexed trace biodetection.

    PubMed

    Chen, Lei; Li, Xiaomin; Shen, Dengke; Zhou, Lei; Zhu, Dan; Fan, Chunhai; Zhang, Fan

    2015-06-01

    Multiplexed detection technology has been attractive for its simultaneous assay of several analytes, which play significant roles in applications such as screening for combinatorial chemistry, genetic analysis, and clinical diagnostics. This work reports a novel and potentially powerful encoding system based upon dispersible suspension arrays of multilayer rare earth core/shell nanoparticles that are capable of multiplexed, high-sensitivity reporting for biomolecule detection by the Z-contrast imaging. These nanobarcode arrays are encoded by nanostructure design based on different atomic numbers. With the well-resolved high-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM) decoding technique, many thousands of unique nanobarcodes can be identified by multilayer core/shell nanostructure. Their applications to multiplexed biodetection of DNA demonstrated the highly sensitive (picomole) features of this novel nanobarcode system.

  8. Development and Validation of a Quantitative, One-Step, Multiplex, Real-Time Reverse Transcriptase PCR Assay for Detection of Dengue and Chikungunya Viruses.

    PubMed

    Simmons, Monika; Myers, Todd; Guevara, Carolina; Jungkind, Donald; Williams, Maya; Houng, Huo-Shu

    2016-07-01

    Dengue virus (DENV) and chikungunya virus (CHIKV) are important human pathogens with common transmission vectors and similar clinical presentations. Patient care may be impacted by the misdiagnosis of DENV and CHIKV in areas where both viruses cocirculate. In this study, we have developed and validated a one-step multiplex reverse transcriptase PCR (RT-PCR) to simultaneously detect, quantify, and differentiate between four DENV serotypes (pan-DENV) and chikungunya virus. The assay uses TaqMan technology, employing two forward primers, three reverse primers, and four fluorophore-labeled probes in a single-reaction format. Coextracted and coamplified RNA was used as an internal control (IC), and in vitro-transcribed DENV and CHIKV RNAs were used to generate standard curves for absolute quantification. The diagnostic 95% limits of detection (LOD) within the linear range were 50 and 60 RNA copies/reaction for DENV (serotypes 1 to 4) and CHIKV, respectively. Our assay was able to detect 53 different strains of DENV, representing four serotypes, and six strains of CHIKV. No cross-reactivity was observed with related flaviviruses and alphaviruses, To evaluate diagnostic sensitivity and specificity, 89 clinical samples positive or negative for DENV (serotypes 1 to 4) and CHIKV by the standard virus isolation method were tested in our assay. The multiplex RT-PCR assay showed 95% sensitivity and 100% specificity for DENV and 100% sensitivity and specificity for CHIKV. With an assay turnaround time of less than 2 h, including extraction of RNA, the multiplex quantitative RT-PCR assay provides rapid diagnosis for the differential detection of two clinically indistinguishable diseases, whose geographical occurrence is increasingly overlapping. PMID:27098955

  9. Matrix Metalloproteinase 2-sensitive Multifunctional Polymeric Micelles for Tumor-specific Co-delivery of siRNA and Hydrophobic Drugs

    PubMed Central

    Zhu, Lin; Perche, Federico; Wang, Tao; Torchilin, Vladimir P

    2014-01-01

    Co-delivery of hydrophilic siRNA and hydrophobic drugs is one of the major challenges for nanomaterial-based medicine. Here, we present a simple but multifunctional micellar platform constructed by a matrix metalloproteinase 2 (MMP2)-sensitive copolymer (PEG-pp-PEI-PE) via self-assembly for tumor-targeted siRNA and drug co-delivery. The micellar nanocarrier possesses several key features for siRNA and drug delivery, including (i) excellent stability; (ii) efficient siRNA condensation by PEI; (iii) hydrophobic drug solubilization in the lipid “core”; (iv) passive tumor targeting via the enhanced permeability and retention (EPR) effect; (v) tumor targeting triggered by the up-regulated tumoral MMP2; and (vi) enhanced cell internalization after MMP2-activated exposure of the previously hidden PEI. These cooperative functions ensure the improved tumor targetability, enhanced tumor cell internalization, and synergistic antitumor activity of co-loaded siRNA and drug. PMID:24529391

  10. Method for multiplex cellular detection of mRNAs using quantum dot fluorescent in situ hybridization.

    PubMed

    Chan, PokMan; Yuen, Tony; Ruf, Frederique; Gonzalez-Maeso, Javier; Sealfon, Stuart C

    2005-10-13

    The photostability and narrow emission spectra of non-organic quantum dot fluorophores (QDs) make them desirable candidates for fluorescent in situ hybridization (FISH) to study the expression of specific mRNA transcripts. We developed a novel method for direct QD labeling of modified oligonucleotide probes through streptavidin and biotin interactions, as well as protocols for their use in multiple-label FISH. We validated this technique in mouse brainstem sections. The subcellular localization of the vesicular monoamine transporter (Vmat2) mRNA corresponds when using probes labeled with two different QDs in the same hybridization. We developed protocols for combined direct QD FISH and QD immunohistochemical labeling within the same neurons as well as for simultaneous study of the subcellular distribution of multiple mRNA targets. We demonstrated increased sensitivity of FISH using QDs in comparison with organic fluorophores. These techniques gave excellent histological results both for multiplex FISH and combined FISH and immunohistochemistry. This approach can facilitate the ultrasensitive simultaneous study of multiple mRNA and protein markers in tissue culture and histological section.

  11. Suppressor analysis of temperature-sensitive mutations of the largest subunit of RNA polymerase I in Saccharomyces cerevisiae: a suppressor gene encodes the second-largest subunit of RNA polymerase I.

    PubMed Central

    Yano, R; Nomura, M

    1991-01-01

    The SRP3-1 mutation is an allele-specific suppressor of temperature-sensitive mutations in the largest subunit (A190) of RNA polymerase I from Saccharomyces cerevisiae. Two mutations known to be suppressed by SRP3-1 are in the putative zinc-binding domain of A190. We have cloned the SRP3 gene by using its suppressor activity and determined its complete nucleotide sequence. We conclude from the following evidence that the SRP3 gene encodes the second-largest subunit (A135) of RNA polymerase I. First, the deduced amino acid sequence of the gene product contains several regions with high homology to the corresponding regions of the second-largest subunits of RNA polymerases of various origins, including those of RNA polymerase II and III from S. cerevisiae. Second, the deduced amino acid sequence contains known amino acid sequences of two tryptic peptides from the A135 subunit of RNA polymerase I purified from S. cerevisiae. Finally, a strain was constructed in which transcription of the SRP3 gene was controlled by the inducible GAL7 promoter. When this strain, which can grow on galactose but not on glucose, was shifted from galactose medium to glucose medium, a large decrease in the cellular concentration of A135 was observed by Western blot analysis. We have also identified the specific amino acid alteration responsible for suppression by SRP3-1 and found that it is located within the putative zinc-binding domain conserved among the second-largest subunits of eucaryotic RNA polymerases. From these results, it is suggested that this putative zinc-binding domain is in physical proximity to and interacts with the putative zinc-binding domain of the A190 subunit. Images PMID:1990281

  12. Multiplex PCR: Optimization and Application in Diagnostic Virology

    PubMed Central

    Elnifro, Elfath M.; Ashshi, Ahmed M.; Cooper, Robert J.; Klapper, Paul E.

    2000-01-01

    PCR has revolutionized the field of infectious disease diagnosis. To overcome the inherent disadvantage of cost and to improve the diagnostic capacity of the test, multiplex PCR, a variant of the test in which more than one target sequence is amplified using more than one pair of primers, has been developed. Multiplex PCRs to detect viral, bacterial, and/or other infectious agents in one reaction tube have been described. Early studies highlighted the obstacles that can jeopardize the production of sensitive and specific multiplex assays, but more recent studies have provided systematic protocols and technical improvements for simple test design. The most useful of these are the empirical choice of oligonucleotide primers and the use of hot start-based PCR methodology. These advances along with others to enhance sensitivity and specificity and to facilitate automation have resulted in the appearance of numerous publications regarding the application of multiplex PCR in the diagnosis of infectious agents, especially those which target viral nucleic acids. This article reviews the principles, optimization, and application of multiplex PCR for the detection of viruses of clinical and epidemiological importance. PMID:11023957

  13. An ultraviolet-sensitive maternal mRNA encoding a cytoskeletal protein may be involved in axis formation in the ascidian embryo

    SciTech Connect

    Jeffery, W.R. )

    1990-09-01

    Ultraviolet (uv) irradiation of the vegetal hemisphere of fertilized eggs during ooplasmic segregation inhibits subsequent gastrulation and axis formation in ascidian embryos. The molecular basis of this phenomenon was investigated in by comparing in vivo protein synthesis and in vitro mRNA translation in normal and uv-irradiated embryos of the ascidian Styela clava. Analysis of protein synthesis by (35S)methionine incorporation, two-dimensional (2D) gel electrophoresis, and autoradiography showed that only 21 of 433 labeled polypeptides were missing or decreased in labeling intensity in uv-irradiated embryos. The most prominent of these was a 30,000 molecular weight (pI 6.0) polypeptide (p30). Extraction of gastrulae with the nonionic detergent Triton X-100 showed that p30 is retained in the detergent insoluble residue, suggesting that it is associated with the cytoskeleton. Several lines of evidence suggest that p30 may be involved in axis formation. First, p30 labeling peaks during gastrulation, when the embryonic axis is being established. Second, axis formation and p30 labeling are abolished by the same threshold uv dose, which is distinct from that required to inactivate muscle cell development. Third, the uv sensitivity period for abolishing p30 labeling and axis formation are both restricted to ooplasmic segregation. In vitro translation of egg RNA followed by 2D gel electrophoresis and autoradiography of the protein products showed that p30 is encoded by a maternal mRNA. The translation of p30 mRNA was abolished by uv irradiation of fertilized eggs during ooplasmic segregation suggesting that this message is a uv-sensitive target. The results are consistent with the hypothesis that uv irradiation blocks gastrulation and axis formation by inhibiting the translation of maternal mRNA localized in the vegetal hemisphere of the fertilized egg.

  14. Simultaneous measurement of multiple ear proteins with multiplex ELISA assays.

    PubMed

    Trune, Dennis R; Larrain, Barbara E; Hausman, Frances A; Kempton, J Beth; MacArthur, Carol J

    2011-05-01

    A recent advancement in enzyme-linked immunosorbent assay (ELISA) technology is the multiplex antibody array that measures multiple proteins simultaneously within a single sample. This allows reduction in sample volume, time, labor, and material costs, while increasing sensitivity over single ELISA. Current multiplex platforms include planar-based systems using microplates or slides, or bead-based suspension assay with microspheres. To determine the applicability of this technology for ear research, we used 3 different multiplex ELISA-based immunoassay arrays from 4 different companies to measure cytokine levels in mouse middle and inner ear tissue lysate extracts 24 h following transtympanic Haemophilus influenzae inoculation. Middle and inner ear tissue lysates were analyzed using testing services from Quansys Biosciences, Aushon Biosystems SearchLight (both microplate-based), MILLIPLEX MAP Sample (bead-based), and a RayBiotech, Inc (slide-based) kit. Samples were assayed in duplicate or triplicate. Results were compared to determine their relative sensitivity and reliability for measures of cytokines related to inflammation. The cytokine pg/ml amounts varied among the multiplex assays, so a comparison also was made of the mean fold increase in cytokines from untreated controls. Several cytokines and chemokines were elevated, the extent dependent upon the assay sensitivity. Those most significantly elevated were IL-1α, IL-1β, IL-6, TNFα, VEGF, and IL-8/MIP-2. The results of the multiplex systems were compared with single ELISA kits (IL-1β, IL-6) to assess sensitivity over the traditional method. Overall, the Quansys Biosciences and SearchLight arrays showed the greatest sensitivity, both employing the same multiplex methodology of a spotted array within a microplate well with chemiluminescent detection. They also were more sensitive than the traditional single ELISA performed with commercial kits and matched gene expression changes determined by quantitative

  15. Delayed Correlation of mRNA and Protein Expression in Rapamycin-treated Cells and a Role for Ggc1 in Cellular Sensitivity to Rapamycin*

    PubMed Central

    Fournier, Marjorie L.; Paulson, Ariel; Pavelka, Norman; Mosley, Amber L.; Gaudenz, Karin; Bradford, William D.; Glynn, Earl; Li, Hua; Sardiu, Mihaela E.; Fleharty, Brian; Seidel, Christopher; Florens, Laurence; Washburn, Michael P.

    2010-01-01

    To identify new molecular targets of rapamycin, an anticancer and immunosuppressive drug, we analyzed temporal changes in yeast over 6 h in response to rapamycin at the transcriptome and proteome levels and integrated the expression patterns with functional profiling. We show that the integration of transcriptomics, proteomics, and functional data sets provides novel insights into the molecular mechanisms of rapamycin action. We first observed a temporal delay in the correlation of mRNA and protein expression where mRNA expression at 1 and 2 h correlated best with protein expression changes after 6 h of rapamycin treatment. This was especially the case for the inhibition of ribosome biogenesis and induction of heat shock and autophagy essential to promote the cellular sensitivity to rapamycin. However, increased levels of vacuolar protease could enhance resistance to rapamycin. Of the 85 proteins identified as statistically significantly changing in abundance, most of the proteins that decreased in abundance were correlated with a decrease in mRNA expression. However, of the 56 proteins increasing in abundance, 26 were not correlated with an increase in mRNA expression. These protein changes were correlated with unchanged or down-regulated mRNA expression. These proteins, involved in mitochondrial genome maintenance, endocytosis, or drug export, represent new candidates effecting rapamycin action whose expression might be post-transcriptionally or post-translationally regulated. We identified GGC1, a mitochondrial GTP/GDP carrier, as a new component of the rapamycin/target of rapamycin (TOR) signaling pathway. We determined that the protein product of GGC1 was stabilized in the presence of rapamycin, and the deletion of the GGC1 enhanced growth fitness in the presence of rapamycin. A dynamic mRNA expression analysis of Δggc1 and wild-type cells treated with rapamycin revealed a key role for Ggc1p in the regulation of ribosome biogenesis and cell cycle progression

  16. Multiplex Immunoassays: Chips and Beads

    PubMed Central

    2010-01-01

    Multiplex analysis is intended to simultaneously look for multiple targets in one sample. This approach has been largely adopted in genomics and progressively expands to various domains of laboratory investigation. In protein analysis, immunoassays are the fundamental methods and their multiplexing and miniaturization is of great applicability to both basic and applied research. Furthermore, the potential of these high-throughput methodologies can be foreseen in the field of clinical diagnostics. The following text describes planar and bead-based arrays, two main strategies of immunoassay multiplexing. Principles, detection methods and strengths of each are shortly discussed. Finally, we mention several challenges linked with the integration of these methods to diagnostics.

  17. Multiplex Touchdown PCR for Rapid Typing of the Opportunistic Pathogen Propionibacterium acnes

    PubMed Central

    Barnard, Emma; Nagy, István; Hunyadkürti, Judit; Patrick, Sheila

    2015-01-01

    The opportunistic human pathogen Propionibacterium acnes is composed of a number of distinct phylogroups, designated types IA1, IA2, IB, IC, II, and III, which vary in their production of putative virulence factors, their inflammatory potential, and their biochemical, aggregative, and morphological characteristics. Although multilocus sequence typing (MLST) currently represents the gold standard for unambiguous phylogroup classification and individual strain identification, it is a labor-intensive and time-consuming technique. As a consequence, we developed a multiplex touchdown PCR assay that in a single reaction can confirm the species identity and phylogeny of an isolate based on its pattern of reaction with six primer sets that target the 16S rRNA gene (all isolates), ATPase (types IA1, IA2, and IC), sodA (types IA2 and IB), atpD (type II), and recA (type III) housekeeping genes, as well as a Fic family toxin gene (type IC). When applied to 312 P. acnes isolates previously characterized by MLST and representing types IA1 (n = 145), IA2 (n = 20), IB (n = 65), IC (n = 7), II (n = 45), and III (n = 30), the multiplex displayed 100% sensitivity and 100% specificity for detecting isolates within each targeted phylogroup. No cross-reactivity with isolates from other bacterial species was observed. This multiplex assay will provide researchers with a rapid, high-throughput, and technically undemanding typing method for epidemiological and phylogenetic investigations. It will facilitate studies investigating the association of lineages with various infections and clinical conditions, and it will serve as a prescreening tool to maximize the number of genetically diverse isolates selected for downstream higher-resolution sequence-based analyses. PMID:25631794

  18. Multiplex PCR for detection of Trypanosoma evansi and Theileria equi in equids of Punjab, India.

    PubMed

    Sumbria, Deepak; Singla, L D; Sharma, Amrita; Bal, M S; Kumar, Sanjay

    2015-07-30

    Multiplex PCR for simultaneous detection of Trypanosoma evansi and Theileria equi in single-step reaction was optimized and employed on 108 equids (99 horses and 9 donkeys/mules) blood samples collected from two agro-climatic zones (Sub-mountain undulating zone and Undulating plain zone) of Punjab to evaluate the status of concurrent infection and associated risk factors. The amplification products of 257 and 709 bp targeting repetitive nucleotide sequence of variable surface glycoproteins of T. evansi and 18S rRNA gene of T. equi, respectively expressed high fidelity of the primer pairs with sequence homology to neighboring geographic isolates. The overall prevalence of T. evansi and T. equi was 3.7 and 1.85%, with Undulating plain zone at higher infection risk for T. equi (OR=3.24, 95% CI=0.28-83.65); and Sub-mountain undulating zone (OR=∞, 95% CI=0.25-∞) for T. evansi. Multiplex PCR revealed higher risk of infection of both T. equi (OR=6.75, 95% CI=0.58-175.38) and T. evansi (OR=2.11, 95% CI=0.05-80.36) in the farms with inappropriate management system. The risk factor associated with the type of host species had an odds ratio of 12.35 (95% CI=0.29-508.37) for donkeys/mules versus horses for T. evansi infection. This group was also at higher risk of infection with Odds ratio (OR) of 4 (95% CI=0.14-53.99) for T. equi. The current investigation brings out various commodities at risk of infection pertaining to equid trypanosomosis and theileriosis evaluated by a rapid and sensitive multiplex PCR assay.

  19. miR-33-5p, a novel mechano-sensitive microRNA promotes osteoblast differentiation by targeting Hmga2

    PubMed Central

    Wang, Han; Sun, Zhongyang; Wang, Yixuan; Hu, Zebing; Zhou, Hua; Zhang, Lianchang; Hong, Bo; Zhang, Shu; Cao, Xinsheng

    2016-01-01

    MicroRNAs (miRNAs) interfere with the translation of specific target mRNAs and are thought to thereby regulate many cellular processes. However, the role of miRNAs in osteoblast mechanotransduction remains to be defined. In this study, we investigated the ability of a miRNA to respond to different mechanical environments and regulate mechano-induced osteoblast differentiation. First, we demonstrated that miR-33-5p expressed by osteoblasts is sensitive to multiple mechanical environments, microgravity and fluid shear stress. We then confirmed the ability of miR-33-5p to promote osteoblast differentiation. Microgravity or fluid shear stress influences osteoblast differentiation partially via miR-33-5p. Through bioinformatics analysis and a luciferase assay, we subsequently confirmed that Hmga2 is a target gene of miR-33-5p that negatively regulates osteoblast differentiation. Moreover, miR-33-5p regulates osteoblast differentiation partially via Hmga2. In summary, our findings demonstrate that miR-33-5p is a novel mechano-sensitive miRNA that can promote osteoblast differentiation and participate in the regulation of differentiation induced by changes in the mechanical environment, suggesting this miRNA as a potential target for the treatment of pathological bone loss. PMID:26980276

  20. A 128 Multiplexing Factor Time-Domain SQUID Multiplexer

    NASA Astrophysics Data System (ADS)

    Prêle, D.; Voisin, F.; Piat, M.; Decourcelle, T.; Perbost, C.; Chapron, C.; Rambaud, D.; Maestre, S.; Marty, W.; Montier, L.

    2016-07-01

    A cryogenic 128:1 Time-Domain Multiplexer (TDM) has been developed for the readout of kilo-pixel Transition Edge Sensor (TES) arrays dedicated to the Q&U Bolometric Interferometer for Cosmology (QUBIC) instrument which aims to measure the B-mode polarization of the Cosmic Microwave Background. Superconducting QUantum Interference Devices (SQUIDs) are usually used to read out TESs. Moreover, SQUIDs are used to build TDM by biasing sequentially the SQUIDs connected together—one for each TES. In addition to this common technique which allows a typical 32 multiplexing factor, a cryogenic integrated circuit provides a 4:1 second multiplexing stage. This cryogenic integrated circuit is one of the original part of our TDM achieving an unprecedented 128 multiplexing factor. We present these two dimension TDM stages: topology of the SQUID multiplexer, operation of the cryogenic integrated circuit, and integration of the full system to read out a TES array dedicated to the QUBIC instrument. Flux-locked loop operation in multiplexed mode is also discussed.

  1. NCLscan: accurate identification of non-co-linear transcripts (fusion, trans-splicing and circular RNA) with a good balance between sensitivity and precision

    PubMed Central

    Chuang, Trees-Juen; Wu, Chan-Shuo; Chen, Chia-Ying; Hung, Li-Yuan; Chiang, Tai-Wei; Yang, Min-Yu

    2016-01-01

    Analysis of RNA-seq data often detects numerous ‘non-co-linear’ (NCL) transcripts, which comprised sequence segments that are topologically inconsistent with their corresponding DNA sequences in the reference genome. However, detection of NCL transcripts involves two major challenges: removal of false positives arising from alignment artifacts and discrimination between different types of NCL transcripts (trans-spliced, circular or fusion transcripts). Here, we developed a new NCL-transcript-detecting method (‘NCLscan’), which utilized a stepwise alignment strategy to almost completely eliminate false calls (>98% precision) without sacrificing true positives, enabling NCLscan outperform 18 other publicly-available tools (including fusion- and circular-RNA-detecting tools) in terms of sensitivity and precision, regardless of the generation strategy of simulated dataset, type of intragenic or intergenic NCL event, read depth of coverage, read length or expression level of NCL transcript. With the high accuracy, NCLscan was applied to distinguishing between trans-spliced, circular and fusion transcripts on the basis of poly(A)- and nonpoly(A)-selected RNA-seq data. We showed that circular RNAs were expressed more ubiquitously, more abundantly and less cell type-specifically than trans-spliced and fusion transcripts. Our study thus describes a robust pipeline for the discovery of NCL transcripts, and sheds light on the fundamental biology of these non-canonical RNA events in human transcriptome. PMID:26442529

  2. A Novel Multiplex PCR Discriminates Bacillus anthracis and Its Genetically Related Strains from Other Bacillus cereus Group Species

    PubMed Central

    Ogawa, Hirohito; Fujikura, Daisuke; Ohnuma, Miyuki; Ohnishi, Naomi; Hang'ombe, Bernard M.; Mimuro, Hitomi; Ezaki, Takayuki; Mweene, Aaron S.; Higashi, Hideaki

    2015-01-01

    Anthrax is an important zoonotic disease worldwide that is caused by Bacillus anthracis, a spore-forming pathogenic bacterium. A rapid and sensitive method to detect B. anthracis is important for anthrax risk management and control in animal cases to address public health issues. However, it has recently become difficult to identify B. anthracis by using previously reported molecular-based methods because of the emergence of B. cereus, which causes severe extra-intestinal infection, as well as the human pathogenic B. thuringiensis, both of which are genetically related to B. anthracis. The close genetic relation of chromosomal backgrounds has led to complexity of molecular-based diagnosis. In this study, we established a B. anthracis multiplex PCR that can screen for the presence of B. anthracis virulent plasmids and differentiate B. anthracis and its genetically related strains from other B. cereus group species. Six sets of primers targeting a chromosome of B. anthracis and B. anthracis-like strains, two virulent plasmids, pXO1 and pXO2, a bacterial gene, 16S rRNA gene, and a mammalian gene, actin-beta gene, were designed. The multiplex PCR detected approximately 3.0 CFU of B. anthracis DNA per PCR reaction and was sensitive to B. anthracis. The internal control primers also detected all bacterial and mammalian DNAs examined, indicating the practical applicability of this assay as it enables monitoring of appropriate amplification. The assay was also applied for detection of clinical strains genetically related to B. anthracis, which were B. cereus strains isolated from outbreaks of hospital infections in Japan, and field strains isolated in Zambia, and the assay differentiated B. anthracis and its genetically related strains from other B. cereus group strains. Taken together, the results indicate that the newly developed multiplex PCR is a sensitive and practical method for detecting B. anthracis. PMID:25774512

  3. Simultaneous detection of four causal agents of tobacco bushy top disease by a multiplex one-step RT-PCR

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Tobacco bushy top disease is a complex disease caused by mixed infection of Tobacco bushy top virus (TBTV), Tobacco vein distorting virus (TVDV), satellite RNA of TBTV (Sat-TBTV) and Tobacco vein distorting virus associate RNA (TVDVaRNA). A one-tube multiplex reverse transcription-PCR (RT-PCR) assay...

  4. High thickness acrylamide photopolymer for peristrophic multiplexing

    NASA Astrophysics Data System (ADS)

    Ortuño, M.; Fernández, E.; Márquez, A.; Gallego, S.; Neipp, C.; Pascual, I.

    2006-05-01

    The acrylamide photolymers are considered interesting materials for holographic media. They have high diffraction efficiency (ratio of the intensities of the diffracted and the incident beams), an intermediate energetic sensitivity among other materials and post-processing steps are not necessary, therefore the media is not altered. The layers of these materials, about 1 mm thick, are a suitable media for recording many diffraction gratings in the same volume of photopolymer using peristrophic multiplexing technique, with great practical importance in the field of holographic memories type WORM (write once read many). In this work we study the recording of diffraction gratings by peristrophic multiplexing with axis of rotation perpendicular to the recording media. The photopolymer is composed of acrylamide as the polymerizable monomer, triethanolamine as radical generator, yellowish eosin as sensitizer and a binder of polyvinyl alcohol. We analyze the holographic behaviour of the material during recording and reconstruction of diffraction gratings using a continuous Nd:YAG laser (532 nm) at an intensity of 5 mW/cm2 as recording laser. The response of the material is monitored after recording with an He-Ne laser. We study the recording process of unslanted diffraction gratings of 1125 lines/mm. The diffraction efficiency of each hologram is seen to decrease as the number of holograms recorded increases, due to consumption of the available dynamic range, in a constant exposure scheduling. It can be seen that the photopolymer works well with high energy levels, without excessive dispersion of light by noise gratings. In order to homogenize the diffraction efficiency of each hologram we use the method proposed by Pu. This method is designed to share all or part of the avaliable dynamic range of the recording material among the holograms to be multiplexed. Using exposure schedules derived from this method we have used 3 scheduling recordings from the algorithm used

  5. MicroRNA-triggered, cascaded and catalytic self-assembly of functional ``DNAzyme ferris wheel'' nanostructures for highly sensitive colorimetric detection of cancer cells

    NASA Astrophysics Data System (ADS)

    Zhou, Wenjiao; Liang, Wenbin; Li, Xin; Chai, Yaqin; Yuan, Ruo; Xiang, Yun

    2015-05-01

    The construction of DNA nanostructures with various sizes and shapes has significantly advanced during the past three decades, yet the application of these DNA nanostructures for solving real problems is still in the early stage. On the basis of microRNA-triggered, catalytic self-assembly formation of the functional ``DNAzyme ferris wheel'' nanostructures, we show here a new signal amplification platform for highly sensitive, label-free and non-enzyme colorimetric detection of a small number of human prostate cancer cells. The microRNA (miR-141), which is catalytically recycled and reused, triggers isothermal self-assembly of a pre-designed, G-quadruplex sequence containing hairpin DNAs into ``DNAzyme ferris wheel''-like nanostructures (in association with hemin) with horseradish peroxidase mimicking activity. These DNAzyme nanostructures catalyze an intensified color transition of the probe solution for highly sensitive detection of miR-141 down to 0.5 pM with the naked eye, and the monitoring of as low as 283 human prostate cancer cells can also, theoretically, be achieved in a colorimetric approach. The work demonstrated here thus offers new opportunities for the construction of functional DNA nanostructures and for the application of these DNA nanostructures as an effective signal amplification means in the sensitive detection of nucleic acid biomarkers.

  6. Multiplexed FBG strain measurement system

    NASA Astrophysics Data System (ADS)

    Helsztynski, Jerzy; Lewandowski, Lech; Jasiewicz, Wieslaw; Jedrzejewski, Kazimierz P.

    2008-11-01

    The idea and, design and realization of fiber Bragg grating multiplexing system is given. Special Bragg gratings with very long and linear side slopes were practically realized. They were tuned for different wavelengths distanced 9nm in each measurement channel. The system was applied for strain control. Special spectrophotometer with linear photodiode array was made. For flexible distributed multiplexing of many sensor channels fiber-optic switches were used.

  7. Evaluation of 16S rRNA Gene PCR Sensitivity and Specificity for Diagnosis of Prosthetic Joint Infection: a Prospective Multicenter Cross-Sectional Study

    PubMed Central

    Plouzeau, Chloé; Tande, Didier; Léger, Julie; Giraudeau, Bruno; Valentin, Anne Sophie; Jolivet-Gougeon, Anne; Vincent, Pascal; Corvec, Stéphane; Gibaud, Sophie; Juvin, Marie Emmanuelle; Héry-Arnaud, Genevieve; Lemarié, Carole; Kempf, Marie; Bret, Laurent; Quentin, Roland; Coffre, Carine; de Pinieux, Gonzague; Bernard, Louis; Burucoa, Christophe

    2014-01-01

    There is no standard method for the diagnosis of prosthetic joint infection (PJI). The contribution of 16S rRNA gene PCR sequencing on a routine basis remains to be defined. We performed a prospective multicenter study to assess the contributions of 16S rRNA gene assays in PJI diagnosis. Over a 2-year period, all patients suspected to have PJIs and a few uninfected patients undergoing primary arthroplasty (control group) were included. Five perioperative samples per patient were collected for culture and 16S rRNA gene PCR sequencing and one for histological examination. Three multicenter quality control assays were performed with both DNA extracts and crushed samples. The diagnosis of PJI was based on clinical, bacteriological, and histological criteria, according to Infectious Diseases Society of America guidelines. A molecular diagnosis was modeled on the bacteriological criterion (≥1 positive sample for strict pathogens and ≥2 for commensal skin flora). Molecular data were analyzed according to the diagnosis of PJI. Between December 2010 and March 2012, 264 suspected cases of PJI and 35 control cases were included. PJI was confirmed in 215/264 suspected cases, 192 (89%) with a bacteriological criterion. The PJIs were monomicrobial (163 cases [85%]; staphylococci, n = 108; streptococci, n = 22; Gram-negative bacilli, n = 16; anaerobes, n = 13; others, n = 4) or polymicrobial (29 cases [15%]). The molecular diagnosis was positive in 151/215 confirmed cases of PJI (143 cases with bacteriological PJI documentation and 8 treated cases without bacteriological documentation) and in 2/49 cases without confirmed PJI (sensitivity, 73.3%; specificity, 95.5%). The 16S rRNA gene PCR assay showed a lack of sensitivity in the diagnosis of PJI on a multicenter routine basis. PMID:25056331

  8. Evaluation of 16S rRNA gene PCR sensitivity and specificity for diagnosis of prosthetic joint infection: a prospective multicenter cross-sectional study.

    PubMed

    Bémer, Pascale; Plouzeau, Chloé; Tande, Didier; Léger, Julie; Giraudeau, Bruno; Valentin, Anne Sophie; Jolivet-Gougeon, Anne; Vincent, Pascal; Corvec, Stéphane; Gibaud, Sophie; Juvin, Marie Emmanuelle; Héry-Arnaud, Genevieve; Lemarié, Carole; Kempf, Marie; Bret, Laurent; Quentin, Roland; Coffre, Carine; de Pinieux, Gonzague; Bernard, Louis; Burucoa, Christophe

    2014-10-01

    There is no standard method for the diagnosis of prosthetic joint infection (PJI). The contribution of 16S rRNA gene PCR sequencing on a routine basis remains to be defined. We performed a prospective multicenter study to assess the contributions of 16S rRNA gene assays in PJI diagnosis. Over a 2-year period, all patients suspected to have PJIs and a few uninfected patients undergoing primary arthroplasty (control group) were included. Five perioperative samples per patient were collected for culture and 16S rRNA gene PCR sequencing and one for histological examination. Three multicenter quality control assays were performed with both DNA extracts and crushed samples. The diagnosis of PJI was based on clinical, bacteriological, and histological criteria, according to Infectious Diseases Society of America guidelines. A molecular diagnosis was modeled on the bacteriological criterion (≥ 1 positive sample for strict pathogens and ≥ 2 for commensal skin flora). Molecular data were analyzed according to the diagnosis of PJI. Between December 2010 and March 2012, 264 suspected cases of PJI and 35 control cases were included. PJI was confirmed in 215/264 suspected cases, 192 (89%) with a bacteriological criterion. The PJIs were monomicrobial (163 cases [85%]; staphylococci, n = 108; streptococci, n = 22; Gram-negative bacilli, n = 16; anaerobes, n = 13; others, n = 4) or polymicrobial (29 cases [15%]). The molecular diagnosis was positive in 151/215 confirmed cases of PJI (143 cases with bacteriological PJI documentation and 8 treated cases without bacteriological documentation) and in 2/49 cases without confirmed PJI (sensitivity, 73.3%; specificity, 95.5%). The 16S rRNA gene PCR assay showed a lack of sensitivity in the diagnosis of PJI on a multicenter routine basis.

  9. Evaluation of 16S rRNA gene PCR sensitivity and specificity for diagnosis of prosthetic joint infection: a prospective multicenter cross-sectional study.

    PubMed

    Bémer, Pascale; Plouzeau, Chloé; Tande, Didier; Léger, Julie; Giraudeau, Bruno; Valentin, Anne Sophie; Jolivet-Gougeon, Anne; Vincent, Pascal; Corvec, Stéphane; Gibaud, Sophie; Juvin, Marie Emmanuelle; Héry-Arnaud, Genevieve; Lemarié, Carole; Kempf, Marie; Bret, Laurent; Quentin, Roland; Coffre, Carine; de Pinieux, Gonzague; Bernard, Louis; Burucoa, Christophe

    2014-10-01

    There is no standard method for the diagnosis of prosthetic joint infection (PJI). The contribution of 16S rRNA gene PCR sequencing on a routine basis remains to be defined. We performed a prospective multicenter study to assess the contributions of 16S rRNA gene assays in PJI diagnosis. Over a 2-year period, all patients suspected to have PJIs and a few uninfected patients undergoing primary arthroplasty (control group) were included. Five perioperative samples per patient were collected for culture and 16S rRNA gene PCR sequencing and one for histological examination. Three multicenter quality control assays were performed with both DNA extracts and crushed samples. The diagnosis of PJI was based on clinical, bacteriological, and histological criteria, according to Infectious Diseases Society of America guidelines. A molecular diagnosis was modeled on the bacteriological criterion (≥ 1 positive sample for strict pathogens and ≥ 2 for commensal skin flora). Molecular data were analyzed according to the diagnosis of PJI. Between December 2010 and March 2012, 264 suspected cases of PJI and 35 control cases were included. PJI was confirmed in 215/264 suspected cases, 192 (89%) with a bacteriological criterion. The PJIs were monomicrobial (163 cases [85%]; staphylococci, n = 108; streptococci, n = 22; Gram-negative bacilli, n = 16; anaerobes, n = 13; others, n = 4) or polymicrobial (29 cases [15%]). The molecular diagnosis was positive in 151/215 confirmed cases of PJI (143 cases with bacteriological PJI documentation and 8 treated cases without bacteriological documentation) and in 2/49 cases without confirmed PJI (sensitivity, 73.3%; specificity, 95.5%). The 16S rRNA gene PCR assay showed a lack of sensitivity in the diagnosis of PJI on a multicenter routine basis. PMID:25056331

  10. Patterned Plasmonic Nanoparticle Arrays for Microfluidic and Multiplexed Biological Assays.

    PubMed

    He, Jie; Boegli, Michelle; Bruzas, Ian; Lum, William; Sagle, Laura

    2015-11-17

    For applications ranging from medical diagnostics and drug screening to chemical and biological warfare detection, inexpensive, rapid-readout, portable devices are required. Localized surface plasmon resonance (LSPR) technologies show substantial promise toward meeting these goals, but the generation of portable, multiplexed and/or microfluidic devices incorporating sensitive nanoparticle arrays is only in its infancy. Herein, we have combined photolithography with Hole Mask Colloidal lithography to pattern uniform nanoparticle arrays for both microfluidic and multiplexed devices. The first proof-of-concept study is carried out with 5- and 7-channel microfluidic devices to acquire one-shot binding curves and protein binding kinetic data. The second proof-of-concept study involved the fabrication of a 96-spot plate that can be inserted into a standard plate reader for the multiplexed detection of protein binding. This versatile fabrication technique should prove useful in next generation chips for bioassays and genetic screening. PMID:26494412

  11. Microbial Contaminants of Cord Blood Units Identified by 16S rRNA Sequencing and by API Test System, and Antibiotic Sensitivity Profiling

    PubMed Central

    França, Luís; Simões, Catarina; Taborda, Marco; Diogo, Catarina; da Costa, Milton S.

    2015-01-01

    Over a period of ten months a total of 5618 cord blood units (CBU) were screened for microbial contamination under routine conditions. The antibiotic resistance profile for all isolates was also examined using ATB strips. The detection rate for culture positive units was 7.5%, corresponding to 422 samples.16S rRNA sequence analysis and identification with API test system were used to identify the culturable aerobic, microaerophilic and anaerobic bacteria from CBUs. From these samples we recovered 485 isolates (84 operational taxonomic units, OTUs) assigned to the classes Bacteroidia, Actinobacteria, Clostridia, Bacilli, Betaproteobacteria and primarily to the Gammaproteobacteria. Sixty-nine OTUs, corresponding to 447 isolates, showed 16S rRNA sequence similarities above 99.0% with known cultured bacteria. However, 14 OTUs had 16S rRNA sequence similarities between 95 and 99% in support of genus level identification and one OTU with 16S rRNA sequence similarity of 90.3% supporting a family level identification only. The phenotypic identification formed 29 OTUs that could be identified to the species level and 9 OTUs that could be identified to the genus level by API test system. We failed to obtain identification for 14 OTUs, while 32 OTUs comprised organisms producing mixed identifications. Forty-two OTUs covered species not included in the API system databases. The API test system Rapid ID 32 Strep and Rapid ID 32 E showed the highest proportion of identifications to the species level, the lowest ratio of unidentified results and the highest agreement to the results of 16S rRNA assignments. Isolates affiliated to the Bacilli and Bacteroidia showed the highest antibiotic multi-resistance indices and microorganisms of the Clostridia displayed the most antibiotic sensitive phenotypes. PMID:26512991

  12. Microbial Contaminants of Cord Blood Units Identified by 16S rRNA Sequencing and by API Test System, and Antibiotic Sensitivity Profiling.

    PubMed

    França, Luís; Simões, Catarina; Taborda, Marco; Diogo, Catarina; da Costa, Milton S

    2015-01-01

    Over a period of ten months a total of 5618 cord blood units (CBU) were screened for microbial contamination under routine conditions. The antibiotic resistance profile for all isolates was also examined using ATB strips. The detection rate for culture positive units was 7.5%, corresponding to 422 samples.16S rRNA sequence analysis and identification with API test system were used to identify the culturable aerobic, microaerophilic and anaerobic bacteria from CBUs. From these samples we recovered 485 isolates (84 operational taxonomic units, OTUs) assigned to the classes Bacteroidia, Actinobacteria, Clostridia, Bacilli, Betaproteobacteria and primarily to the Gammaproteobacteria. Sixty-nine OTUs, corresponding to 447 isolates, showed 16S rRNA sequence similarities above 99.0% with known cultured bacteria. However, 14 OTUs had 16S rRNA sequence similarities between 95 and 99% in support of genus level identification and one OTU with 16S rRNA sequence similarity of 90.3% supporting a family level identification only. The phenotypic identification formed 29 OTUs that could be identified to the species level and 9 OTUs that could be identified to the genus level by API test system. We failed to obtain identification for 14 OTUs, while 32 OTUs comprised organisms producing mixed identifications. Forty-two OTUs covered species not included in the API system databases. The API test system Rapid ID 32 Strep and Rapid ID 32 E showed the highest proportion of identifications to the species level, the lowest ratio of unidentified results and the highest agreement to the results of 16S rRNA assignments. Isolates affiliated to the Bacilli and Bacteroidia showed the highest antibiotic multi-resistance indices and microorganisms of the Clostridia displayed the most antibiotic sensitive phenotypes.

  13. Effect of starvation on activities and mRNA expression of lipoprotein lipase and hormone-sensitive lipase in tilapia (Oreochromis niloticus x O. areus).

    PubMed

    Han, Chunyan; Wen, Xiaobo; Zheng, Qingmei; Li, Haobo

    2011-03-01

    A 4-week study was conducted to determine the effect of starvation on activities and mRNA expression of lipoprotein lipase (LPL) and hormone-sensitive lipase (HSL) in hybrid tilapia (Oreochromis niloticus x O. areus). The tissue samples were sampled once a week. Results showed that body weight (BW) and hepatosomatic index (HSI) were decreased significantly (P < 0.05) during starvation. The percentages of crude fat and crude protein in the whole body and the fat content in muscle decreased significantly (P < 0.05), while the rate of moisture and crude ash increased significantly (P < 0.05). The response of LPL, HSL activities and mRNA expression in tissues was tissue dependent. The activities of LPL and HSL in muscle at day 7 were elevated by 2.5 times (P < 0.05) and 11.8 times (P < 0.05) of the value at day 0, respectively, and both then decreased to pre-starvation levels at day 14 and finally stabilized at a certain level afterward. LPL and HSL mRNA abundance in muscle remained relatively stable between 0 and 14 day; then, a significant increase was seen after 14 days. In the liver, LPL activity maintained a significantly increasing trend during starvation, while HSL activity rose dramatically at day 7 of starvation by 2.35 times (P < 0.05) and finally stabilized at a certain level. The mRNA abundance of liver LPL increased significantly during the whole process of starvation (P < 0.05), whereas the mRNA abundance of liver HSL decreased significantly at day 7 of starvation, elevating significantly afterward (P < 0.05).

  14. Microbial Contaminants of Cord Blood Units Identified by 16S rRNA Sequencing and by API Test System, and Antibiotic Sensitivity Profiling.

    PubMed

    França, Luís; Simões, Catarina; Taborda, Marco; Diogo, Catarina; da Costa, Milton S

    2015-01-01

    Over a period of ten months a total of 5618 cord blood units (CBU) were screened for microbial contamination under routine conditions. The antibiotic resistance profile for all isolates was also examined using ATB strips. The detection rate for culture positive units was 7.5%, corresponding to 422 samples.16S rRNA sequence analysis and identification with API test system were used to identify the culturable aerobic, microaerophilic and anaerobic bacteria from CBUs. From these samples we recovered 485 isolates (84 operational taxonomic units, OTUs) assigned to the classes Bacteroidia, Actinobacteria, Clostridia, Bacilli, Betaproteobacteria and primarily to the Gammaproteobacteria. Sixty-nine OTUs, corresponding to 447 isolates, showed 16S rRNA sequence similarities above 99.0% with known cultured bacteria. However, 14 OTUs had 16S rRNA sequence similarities between 95 and 99% in support of genus level identification and one OTU with 16S rRNA sequence similarity of 90.3% supporting a family level identification only. The phenotypic identification formed 29 OTUs that could be identified to the species level and 9 OTUs that could be identified to the genus level by API test system. We failed to obtain identification for 14 OTUs, while 32 OTUs comprised organisms producing mixed identifications. Forty-two OTUs covered species not included in the API system databases. The API test system Rapid ID 32 Strep and Rapid ID 32 E showed the highest proportion of identifications to the species level, the lowest ratio of unidentified results and the highest agreement to the results of 16S rRNA assignments. Isolates affiliated to the Bacilli and Bacteroidia showed the highest antibiotic multi-resistance indices and microorganisms of the Clostridia displayed the most antibiotic sensitive phenotypes. PMID:26512991

  15. Multiplexed protein profiling by sequential affinity capture

    PubMed Central

    Ayoglu, Burcu; Birgersson, Elin; Mezger, Anja; Nilsson, Mats; Uhlén, Mathias; Nilsson, Peter

    2016-01-01

    Antibody microarrays enable parallelized and miniaturized analysis of clinical samples, and have proven to provide novel insights for the analysis of different proteomes. However, there are concerns that the performance of such direct labeling and single antibody assays are prone to off‐target binding due to the sample context. To improve selectivity and sensitivity while maintaining the possibility to conduct multiplexed protein profiling, we developed a multiplexed and semi‐automated sequential capture assay. This novel bead‐based procedure encompasses a first antigen capture, labeling of captured protein targets on magnetic particles, combinatorial target elution and a read‐out by a secondary capture bead array. We demonstrate in a proof‐of‐concept setting that target detection via two sequential affinity interactions reduced off‐target contribution, while lowered background and noise levels, improved correlation to clinical values compared to single binder assays. We also compared sensitivity levels with single binder and classical sandwich assays, explored the possibility for DNA‐based signal amplification, and demonstrate the applicability of the dual capture bead‐based antibody microarray for biomarker analysis. Hence, the described concept enhances the possibilities for antibody array assays to be utilized for protein profiling in body fluids and beyond. PMID:26935855

  16. A label-free and self-assembled electrochemical biosensor for highly sensitive detection of cyclic diguanylate monophosphate (c-di-GMP) based on RNA riboswitch.

    PubMed

    Xie, Qingyun; Zhao, Fulin; Liu, Hongrui; Shan, Yanke; Liu, Fei

    2015-07-01

    Cyclic diguanylate monophosphate (c-di-GMP) is an important second messenger that regulates a variety of complex physiological processes involved in motility, virulence, biofilm formation and cell cycle progression in several bacteria. Herein we report a simple label-free and self-assembled RNA riboswitch-based biosensor for sensitive and selective detection of c-di-GMP. The detectable concentration range of c-di-GMP is from 50 nM to 1 μM with a detection limit of 50 nM.

  17. Sensitive detection of miRNA by using hybridization chain reaction coupled with positively charged gold nanoparticles.

    PubMed

    Miao, Xiangmin; Ning, Xue; Li, Zongbing; Cheng, Zhiyuan

    2016-01-01

    Positively charged gold nanoparticles (+)AuNPs can adsorb onto the negatively charged surface of single-stranded DNA (ssDNA) or double-stranded DNA (dsDNA). Herein, long-range dsDNA polymers could form based on the hybridization chain reaction (HCR) of two hairpin probes (H1 and H2) by using miRNA-21 as an initiator. (+)AuNPs could adsorb onto the negatively charged surface of such long-range dsDNA polymers based on the electrostatic adsorption, which directly resulted in the precipitation of (+)AuNPs and the decrease of (+)AuNPs absorption spectra. Under optimal conditions, miRNA-21 detection could be realized in the range of 20 pM-10 nM with a detection limit of 6.8 pM. In addition, (+)AuNPs used here are much more stable than commonly used negatively charged gold nanoparticles ((-)AuNPs) in mixed solution that contained salt, protein or other metal ions. Importantly, the assay could realize the detection of miRNA in human serum samples. PMID:27576601

  18. Sensitive detection of miRNA by using hybridization chain reaction coupled with positively charged gold nanoparticles

    PubMed Central

    Miao, Xiangmin; Ning, Xue; Li, Zongbing; Cheng, Zhiyuan

    2016-01-01

    Positively charged gold nanoparticles (+)AuNPs can adsorb onto the negatively charged surface of single-stranded DNA (ssDNA) or double-stranded DNA (dsDNA). Herein, long-range dsDNA polymers could form based on the hybridization chain reaction (HCR) of two hairpin probes (H1 and H2) by using miRNA-21 as an initiator. (+)AuNPs could adsorb onto the negatively charged surface of such long-range dsDNA polymers based on the electrostatic adsorption, which directly resulted in the precipitation of (+)AuNPs and the decrease of (+)AuNPs absorption spectra. Under optimal conditions, miRNA-21 detection could be realized in the range of 20 pM-10 nM with a detection limit of 6.8 pM. In addition, (+)AuNPs used here are much more stable than commonly used negatively charged gold nanoparticles ((−)AuNPs) in mixed solution that contained salt, protein or other metal ions. Importantly, the assay could realize the detection of miRNA in human serum samples. PMID:27576601

  19. Rapid and Sensitive Detection of RNA Viruses Based on Reverse Transcription Loop-Mediated Isothermal Amplification, Magnetic Nanoparticles, and Chemiluminescence.

    PubMed

    Wang, Jiuhai; Lu, Peng; Yan, Jieni; Zhang, Yufan; Huang, Lanye; Ali, Zeeshan; Li, Zhiyang; He, Nongyue

    2016-04-01

    RNA viruses, particularly, the highly pathogenic avian influenza (HPAI) virus, pose serious health concerns, and cause huge economic losses worldwide. Diagnostic tools for the early detection of these deadly RNA viruses are urgently needed to implement treatment and disease control strategies. Conventional reverse transcription polymerase chain reaction (RT-PCR)-based chemiluminescent (RT-PCR-CL) detection is frequently used for the diagnosis of viral infections. However, the requirements for expensive PCR machines and longer thermocycling times are significant drawbacks. In this study, we propose a method based on reverse transcription loop-mediated isothermal amplification (RT-LAMP) combined with chemiluminescence (CL) to detect H7N9 virus. The proposed method does not require any expensive instruments, and processing time is remarkably shortened compared to that of RT-PCR-CL. Since several factors including RT-LAMP temperature, probe concentration, hybridization temperature, and hybridization duration might affect the CL signal, each of these parameters was investigated and optimized. One thousand copies/mL of H7N9 RNA were detectable using the optimized RT-LAMP-CL method. The detection time was significantly reduced by using RT-LAMP, in comparison with conventional RT-PCR-CL. This technique holds great promise for viral detection and diagnosis, especially with regard to avian influenza virus. PMID:27301197

  20. Multiplex component-based allergen microarray in recent clinical studies.

    PubMed

    Patelis, A; Borres, M P; Kober, A; Berthold, M

    2016-08-01

    During the last decades component-resolved diagnostics either as singleplex or multiplex measurements has been introduced into the field of clinical allergology, providing important information that cannot be obtained from extract-based tests. Here we review recent studies that demonstrate clinical applications of the multiplex microarray technique in the diagnosis and risk assessment of allergic patients, and its usefulness in studies of allergic diseases. The usefulness of ImmunoCAP ISAC has been validated in a wide spectrum of allergic diseases like asthma, allergic rhinoconjunctivitis, atopic dermatitis, eosinophilic esophagitis, food allergy and anaphylaxis. ISAC provides a broad picture of a patient's sensitization profile from a single test, and provides information on specific and cross-reactive sensitizations that facilitate diagnosis, risk assessment, and disease management. Furthermore, it can reveal unexpected sensitizations which may explain anaphylaxis previously categorized as idiopathic and also display for the moment clinically non-relevant sensitizations. ISAC can facilitate a better selection of relevant allergens for immunotherapy compared with extract testing. Microarray technique can visualize the allergic march and molecular spreading in the preclinical stages of allergic diseases, and may indicate that the likelihood of developing symptomatic allergy is associated with specific profiles of sensitization to allergen components. ISAC is shown to be a useful tool in routine allergy diagnostics due to its ability to improve risk assessment, to better select relevant allergens for immunotherapy as well as detecting unknown sensitization. Multiplex component testing is especially suitable for patients with complex symptomatology. PMID:27196983

  1. Multiplex PCR for diagnosis of Theileria uilenbergi, Theileria luwenshuni, and Theileria ovis in small ruminants.

    PubMed

    Zhang, Xiao; Liu, Zhijie; Yang, Jifei; Chen, Ze; Guan, Guiquan; Ren, Qiaoyun; Liu, Aihong; Luo, Jianxun; Yin, Hong; Li, Youquan

    2014-02-01

    Infections with Theileria sp. may cause significant economic losses to the sheep industry. Species identification based on microscopic examination is difficult, and more suitable methods are required for the rapid detection and identification of Theileria sp, in clinical specimens. In this study, a multiplex polymerase chain reaction (mPCR) assay was developed to simultaneously identify three individual Theileria species in small ruminants. Three pairs of specific, sensitive primers were designed on the basis of the 5.8S ribosomal RNA gene (Theileria luwenshuni and Theileria ovis) and the 18S ribosomal RNA gene (Theileria uilenbergi) to generate target products of 303, 884, and 530 bp, respectively. Standard DNA for each of the three species was extracted from blood recovered from infected sheep, and a preliminary study was conducted on 56 sheep to verify the reliability of the system. Optimal PCR conditions, including primer concentration, annealing time, and the number of amplification cycles, were established. The assay sensitivity under these conditions was 10(-3) % parasitemia, and its specificity was 100 %. The results of the study suggest that mPCR represents a simple, efficient test method as a practical alternative for the rapid detection and identification of Theileria species in small ruminants.

  2. Analog signal multiplexing for PSAPD-based PET detectors: simulation and experimental validation

    PubMed Central

    Lau, Frances W Y; Vandenbroucke, Arne; Reynolds, Paul D; Olcott, Peter D; Horowitz, Mark A; Levin, Craig S

    2013-01-01

    A 1 mm3 resolution clinical positron emission tomography (PET) system employing 4608 position-sensitive avalanche photodiodes (PSAPDs) is under development. This paper describes a detector multiplexing technique that simplifies the readout electronics and reduces the density of the circuit board design. The multiplexing scheme was validated using a simulation framework that models the PSAPDs and front-end multiplexing circuits to predict the signal-to-noise ratio and flood histogram performance. Two independent experimental setups measured the energy resolution, time resolution, crystal identification ability and count rate both with and without multiplexing. With multiplexing, there was no significant degradation in energy resolution, time resolution and count rate. There was a relative 6.9 ± .0% and 9.4 ± 1.0% degradation in the figure of merit that characterizes the crystal identification ability observed in the measured and simulated ceramic-mounted PSAPD module flood histograms, respectively. PMID:21081831

  3. Efficient exploration of multiplex networks

    NASA Astrophysics Data System (ADS)

    Battiston, Federico; Nicosia, Vincenzo; Latora, Vito

    2016-04-01

    Efficient techniques to navigate networks with local information are fundamental to sample large-scale online social systems and to retrieve resources in peer-to-peer systems. Biased random walks, i.e. walks whose motion is biased on properties of neighbouring nodes, have been largely exploited to design smart local strategies to explore a network, for instance by constructing maximally mixing trajectories or by allowing an almost uniform sampling of the nodes. Here we introduce and study biased random walks on multiplex networks, graphs where the nodes are related through different types of links organised in distinct and interacting layers, and we provide analytical solutions for their long-time properties, including the stationary occupation probability distribution and the entropy rate. We focus on degree-biased random walks and distinguish between two classes of walks, namely those whose transition probability depends on a number of parameters which is extensive in the number of layers, and those whose motion depends on intrinsically multiplex properties of the neighbouring nodes. We analyse the effect of the structure of the multiplex network on the steady-state behaviour of the walkers, and we find that heterogeneous degree distributions as well as the presence of inter-layer degree correlations and edge overlap determine the extent to which a multiplex can be efficiently explored by a biased walk. Finally we show that, in real-world multiplex transportation networks, the trade-off between efficient navigation and resilience to link failure has resulted into systems whose diffusion properties are qualitatively different from those of appropriately randomised multiplex graphs. This fact suggests that multiplexity is an important ingredient to include in the modelling of real-world systems.

  4. Evidence for genetic regulation of mRNA expression of the dosage-sensitive gene retinoic acid induced-1 (RAI1) in human brain

    PubMed Central

    Chen, Li; Tao, Yu; Song, Fan; Yuan, Xi; Wang, Jian; Saffen, David

    2016-01-01

    RAI1 (retinoic acid induced-1) is a dosage-sensitive gene that causes Smith-Magenis syndrome (SMS) when mutated or deleted and Potocki-Lupski Syndrome (PTLS) when duplicated, with psychiatric features commonly observed in both syndromes. How common genetic variants regulate this gene, however, is unknown. In this study, we found that RAI1 mRNA expression in Chinese prefrontal and temporal cortex correlate with genotypes of common single nucleotide polymorphisms (SNPs) located in the RAI1 5′-upstream region. Using genotype imputation, “R2-Δ2” analysis, and data from the RegulomeDB database, we identified SNPs rs4925102 and rs9907986 as possible regulatory variants, accounting for approximately 30–40% of the variance in RAI1 mRNA expression in both brain regions. Specifically, rs4925102 and rs9907986 are predicted to disrupt the binding of retinoic acid RXR-RAR receptors and the transcription factor DEAF1 (Deformed epidermal autoregulatory factor-1), respectively. Consistent with these predictions, we observed binding of RXRα and RARα to the predicted RAI1 target in chromatin immunoprecipitation assays. Retinoic acid is crucial for early development of the central neural system, and DEAF1 is associated with intellectual disability. The observation that a significant portion of RAI1 mRNA expression is genetically controlled raises the possibility that common RAI1 5′-region regulatory variants contribute more generally to psychiatric disorders. PMID:26743651

  5. A temperature-sensitive allele of a putative mRNA splicing helicase down-regulates many cell wall genes and causes radial swelling in Arabidopsis thaliana.

    PubMed

    Howles, Paul A; Gebbie, Leigh K; Collings, David A; Varsani, Arvind; Broad, Ronan C; Ohms, Stephen; Birch, Rosemary J; Cork, Ann H; Arioli, Tony; Williamson, Richard E

    2016-05-01

    The putative RNA helicase encoded by the Arabidopsis gene At1g32490 is a homolog of the yeast splicing RNA helicases Prp2 and Prp22. We isolated a temperature-sensitive allele (rsw12) of the gene in a screen for root radial swelling mutants. Plants containing this allele grown at the restrictive temperature showed weak radial swelling, were stunted with reduced root elongation, and contained reduced levels of cellulose. The role of the protein was further explored by microarray analysis. By using both fold change cutoffs and a weighted gene coexpression network analysis (WGCNA) to investigate coexpression of genes, we found that the radial swelling phenotype was not linked to genes usually associated with primary cell wall biosynthesis. Instead, the mutation has strong effects on expression of secondary cell wall related genes. Many genes potentially associated with secondary walls were present in the most significant WGCNA module, as were genes coding for arabinogalactans and proteins with GPI anchors. The proportion of up-regulated genes that possess introns in rsw12 was above that expected if splicing was unrelated to the activity of the RNA helicase, suggesting that the helicase does indeed play a role in splicing in Arabidopsis. The phenotype may be due to a change in the expression of one or more genes coding for cell wall proteins.

  6. Chemical modification of chitosan with pH-sensitive molecules and specific ligands for efficient DNA transfection and siRNA silencing.

    PubMed

    Singh, Bijay; Choi, Yun-Jaie; Park, In-Kyu; Akaike, Toshihiro; Cho, Chong-Su

    2014-01-01

    Successful gene therapy depends on the development of efficient and cell-specific gene delivery systems. Currently, animal viral vectors have been mostly used for in vivo and in clinical trials owing to their high transduction efficiency. However, they suffer from numerous limitations such as biosafety, immunogenicity, gene packaging capacity, complicated production and cell specificity. Therefore non-viral vectors are attractive alternatives to viral gene delivery systems due to their low toxicity, relatively easy production and greater diversity. Among non-viral vectors, chitosan and chitosan derivatives have been extensively utilized as gene carriers owing to their low immunogenicity, biocompatibility, biodegradability, low toxicity and ease of chemical modifications. However, low transfection efficiency of DNA (or low gene silencing of siRNA) and low cell specificity of chitosan should be overcome before clinical trials. The objective of this review is to summarize several parameters affecting the transfection efficiency of DNA (or gene silencing of siRNA) for the promising use of chitosan as gene carriers. Besides, chemical modifications of chitosan with pH-sensitive molecules and specific ligands so as to enhance the transfection efficiency of DNA (or gene silencing of siRNA) and cell specificity will be covered. PMID:24730283

  7. Defective RNA replication and late gene expression in temperature-sensitive influenza viruses expressing deleted forms of the NS1 protein.

    PubMed

    Falcón, Ana M; Marión, Rosa M; Zürcher, Thomas; Gómez, Paulino; Portela, Agustín; Nieto, Amelia; Ortín, Juan

    2004-04-01

    Influenza A virus mutants expressing C-terminally deleted forms of the NS1 protein (NS1-81 and NS1-110) were generated by plasmid rescue. These viruses were temperature sensitive and showed a small plaque size at the permissive temperature. The accumulation of virion RNA in mutant virus-infected cells was reduced at the restrictive temperature, while the accumulation of cRNA or mRNA was not affected, indicating that the NS1 protein is involved in the control of transcription versus replication processes in the infection. The synthesis and accumulation of late virus proteins were reduced in NS1-81 mutant-infected cells at the permissive temperature and were essentially abolished for both viruses at the restrictive temperature, while synthesis and accumulation of nucleoprotein (NP) were unaffected. Probably as a consequence, the nucleocytoplasmic export of virus NP was strongly inhibited at the restrictive temperature. These results indicate that the NS1 protein is essential for nuclear and cytoplasmic steps during the virus cycle.

  8. Conformation-sensitive nucleoside analogues as topology-specific fluorescence turn-on probes for DNA and RNA G-quadruplexes.

    PubMed

    Tanpure, Arun A; Srivatsan, Seergazhi G

    2015-12-15

    Development of probes that can discriminate G-quadruplex (GQ) structures and indentify efficient GQ binders on the basis of topology and nucleic acid type is highly desired to advance GQ-directed therapeutic strategies. In this context, we describe the development of minimally perturbing and environment-sensitive pyrimidine nucleoside analogues, based on a 5-(benzofuran-2-yl)uracil core, as topology-specific fluorescence turn-on probes for human telomeric DNA and RNA GQs. The pyrimidine residues of one of the loop regions (TTA) of telomeric DNA and RNA GQ oligonucleotide (ON) sequences were replaced with 5-benzofuran-modified 2'-deoxyuridine and uridine analogues. Depending on the position of modification the fluorescent nucleoside analogues distinguish antiparallel, mixed parallel-antiparallel and parallel stranded DNA and RNA GQ topologies from corresponding duplexes with significant enhancement in fluorescence intensity and quantum yield. Further, these GQ sensors enabled the development of a simple fluorescence binding assay to quantify topology- and nucleic acid-specific binding of small molecule ligands to GQ structures. Together, our results demonstrate that these nucleoside analogues are useful GQ probes, which are anticipated to provide new opportunities to study and discover efficient G-quadruplex binders of therapeutic potential.

  9. mRNA expression of diacylglycerol kinase isoforms in insulin-sensitive tissues: effects of obesity and insulin resistance.

    PubMed

    Mannerås-Holm, Louise; Kirchner, Henriette; Björnholm, Marie; Chibalin, Alexander V; Zierath, Juleen R

    2015-04-01

    Diacylglycerol kinase (DGK) isoforms regulate signal transduction and lipid metabolism. DGKδ deficiency leads to hyperglycemia, peripheral insulin resistance, and metabolic inflexibility. Thus, dysregulation of other DGK isoforms may play a role in metabolic dysfunction. We investigated DGK isoform mRNA expression in extensor digitorum longus (EDL) and soleus muscle, liver as well as subcutaneous and epididymal adipose tissue in C57BL/6J mice and obese and insulin-resistant ob/ob mice. All DGK isoforms, except for DGKκ, were detectable, although with varying mRNA expression. Liver DGK expression was generally lowest, with several isoforms undetectable. In soleus muscle, subcutaneous and epididymal adipose tissue, DGKδ was the most abundant isoform. In EDL muscle, DGKα and DGKζ were the most abundant isoforms. In liver, DGKζ was the most abundant isoform. Comparing obese insulin-resistant ob/ob mice to lean C57BL/6J mice, DGKβ, DGKι, and DGKθ were increased and DGKε expression was decreased in EDL muscle, while DGKβ, DGKη and DGKθ were decreased and DGKδ and DGKι were increased in soleus muscle. In liver, DGKδ and DGKζ expression was increased in ob/ob mice. DGKη was increased in subcutaneous fat, while DGKζ was increased and DGKβ, DGKδ, DGKη and DGKε were decreased in epididymal fat from ob/ob mice. In both adipose tissue depots, DGKα and DGKγ were decreased and DGKι was increased in ob/ob mice. In conclusion, DGK mRNA expression is altered in an isoform- and tissue-dependent manner in obese insulin-resistant ob/ob mice. DGK isoforms likely have divergent functional roles in distinct tissues, which may contribute to metabolic dysfunction. PMID:25847921

  10. Research resource: whole transcriptome RNA sequencing detects multiple 1α,25-dihydroxyvitamin D(3)-sensitive metabolic pathways in developing zebrafish.

    PubMed

    Craig, Theodore A; Zhang, Yuji; McNulty, Melissa S; Middha, Sumit; Ketha, Hemamalini; Singh, Ravinder J; Magis, Andrew T; Funk, Cory; Price, Nathan D; Ekker, Stephen C; Kumar, Rajiv

    2012-09-01

    The biological role of vitamin D receptors (VDR), which are abundantly expressed in developing zebrafish (Danio rerio) as early as 48 h after fertilization, and before the development of a mineralized skeleton and mature intestine and kidney, is unknown. We probed the role of VDR in developing zebrafish biology by examining changes in expression of RNA by whole transcriptome shotgun sequencing (RNA-seq) in fish treated with picomolar concentrations of the VDR ligand and hormonal form of vitamin D(3), 1α,25-dihydroxyvitamin D(3) [1α,25(OH)(2)D(3))].We observed significant changes in RNAs of transcription factors, leptin, peptide hormones, and RNAs encoding proteins of fatty acid, amino acid, xenobiotic metabolism, receptor-activator of NFκB ligand (RANKL), and calcitonin-like ligand receptor pathways. Early highly restricted, and subsequent massive changes in more than 10% of expressed cellular RNA were observed. At days post fertilization (dpf) 2 [24 h 1α,25(OH)(2)D(3)-treatment], only four RNAs were differentially expressed (hormone vs. vehicle). On dpf 4 (72 h treatment), 77 RNAs; on dpf 6 (120 h treatment) 1039 RNAs; and on dpf 7 (144 h treatment), 2407 RNAs were differentially expressed in response to 1α,25(OH)(2)D(3). Fewer RNAs (n = 481) were altered in dpf 7 larvae treated for 24 h with 1α,25(OH)(2)D(3) vs. those treated with hormone for 144 h. At dpf 7, in 1α,25(OH)(2)D(3)-treated larvae, pharyngeal cartilage was larger and mineralization was greater. Changes in expression of RNAs for transcription factors, peptide hormones, and RNAs encoding proteins integral to fatty acid, amino acid, leptin, calcitonin-like ligand receptor, RANKL, and xenobiotic metabolism pathways, demonstrate heretofore unrecognized mechanisms by which 1α,25(OH)(2)D(3) functions in vivo in developing eukaryotes.

  11. RapMap: a rapid, sensitive and accurate tool for mapping RNA-seq reads to transcriptomes

    PubMed Central

    Srivastava, Avi; Sarkar, Hirak; Gupta, Nitish; Patro, Rob

    2016-01-01

    Motivation: The alignment of sequencing reads to a transcriptome is a common and important step in many RNA-seq analysis tasks. When aligning RNA-seq reads directly to a transcriptome (as is common in the de novo setting or when a trusted reference annotation is available), care must be taken to report the potentially large number of multi-mapping locations per read. This can pose a substantial computational burden for existing aligners, and can considerably slow downstream analysis. Results: We introduce a novel concept, quasi-mapping, and an efficient algorithm implementing this approach for mapping sequencing reads to a transcriptome. By attempting only to report the potential loci of origin of a sequencing read, and not the base-to-base alignment by which it derives from the reference, RapMap—our tool implementing quasi-mapping—is capable of mapping sequencing reads to a target transcriptome substantially faster than existing alignment tools. The algorithm we use to implement quasi-mapping uses several efficient data structures and takes advantage of the special structure of shared sequence prevalent in transcriptomes to rapidly provide highly-accurate mapping information. We demonstrate how quasi-mapping can be successfully applied to the problems of transcript-level quantification from RNA-seq reads and the clustering of contigs from de novo assembled transcriptomes into biologically meaningful groups. Availability and implementation: RapMap is implemented in C ++11 and is available as open-source software, under GPL v3, at https://github.com/COMBINE-lab/RapMap. Contact: rob.patro@cs.stonybrook.edu Supplementary information: Supplementary data are available at Bioinformatics online. PMID:27307617

  12. A Multiplex PCR Approach for Detecting Dual Infections and Recombinants Involving Major HIV Variants

    PubMed Central

    Cappy, Pierre; De Oliveira, Fabienne; Gueudin, Marie; Alessandri-Gradt, Elodie

    2016-01-01

    The cocirculation of different HIV types and groups can lead to dual infections and recombinants, which hinder diagnosis and therapeutic management. We designed two multiplex PCRs (mPCRs) coupled with capillary electrophoresis to facilitate the detection of such infections. The first, MMO2, targets three variants (HIV-1/M, HIV-1/O, and HIV-2), and the second, MMO, targets HIV-1/M and HIV-1/O. These mPCRs were validated on DNA and RNA extracts from 19 HIV-1/M, 12 HIV-1/O, and 13 HIV-2 cultures and from mixtures simulating dual infections. They were then assessed with DNA and RNA extracts from samples of 47 clinical monoinfections and HIV-1/M+O dual infections or infections with HIV-1/MO recombinants. Both mPCRs had excellent specificity. Sensitivities ranged from 80 to 100% for in vitro samples and from 58 to 100% for clinical samples, with the results obtained depending on the material used and the region of the genome concerned. Sensitivity was generally lower for DNA than for RNA and for amplifications of the integrase and matrix regions. In terms of global detection (at least one target gene for each strain), both mPCRs yielded a detection rate of 100% for in vitro samples. MMO2 detected 100% of the clinical strains from DNA and 97% from RNA, whereas MMO detected 100% of the strains from both materials. Thus, for in vitro and clinical samples, MMO2 was a useful tool for detecting dual infections with HIV-1 and HIV-2 (referred to as HIV-1+HIV-2) and HIV-1/M+O, and MMO was useful for detecting both MO dual infections and MO mosaic patterns. PMID:26912747

  13. Mutations in UVSSA cause UV-sensitive syndrome and impair RNA polymerase IIo processing in transcription-coupled nucleotide-excision repair.

    PubMed

    Nakazawa, Yuka; Sasaki, Kensaku; Mitsutake, Norisato; Matsuse, Michiko; Shimada, Mayuko; Nardo, Tiziana; Takahashi, Yoshito; Ohyama, Kaname; Ito, Kosei; Mishima, Hiroyuki; Nomura, Masayo; Kinoshita, Akira; Ono, Shinji; Takenaka, Katsuya; Masuyama, Ritsuko; Kudo, Takashi; Slor, Hanoch; Utani, Atsushi; Tateishi, Satoshi; Yamashita, Shunichi; Stefanini, Miria; Lehmann, Alan R; Yoshiura, Koh-ichiro; Ogi, Tomoo

    2012-05-01

    UV-sensitive syndrome (UV(S)S) is a genodermatosis characterized by cutaneous photosensitivity without skin carcinoma. Despite mild clinical features, cells from individuals with UV(S)S, like Cockayne syndrome cells, are very UV sensitive and are deficient in transcription-coupled nucleotide-excision repair (TC-NER), which removes DNA damage in actively transcribed genes. Three of the seven known UV(S)S cases carry mutations in the Cockayne syndrome genes ERCC8 or ERCC6 (also known as CSA and CSB, respectively). The remaining four individuals with UVSS , one of whom is described for the first time here, formed a separate UV(S)S-A complementation group; however, the responsible gene was unknown. Using exome sequencing, we determine that mutations in the UVSSA gene (formerly known as KIAA1530) cause UV(S)S-A. The UVSSA protein interacts with TC-NER machinery and stabilizes the ERCC6 complex; it also facilitates ubiquitination of RNA polymerase IIo stalled at DNA damage sites. Our findings provide mechanistic insights into the processing of stalled RNA polymerase and explain the different clinical features across these TC-NER–deficient disorders. PMID:22466610

  14. Multiplexed Western Blotting Using Microchip Electrophoresis.

    PubMed

    Jin, Shi; Furtaw, Michael D; Chen, Huaxian; Lamb, Don T; Ferguson, Stephen A; Arvin, Natalie E; Dawod, Mohamed; Kennedy, Robert T

    2016-07-01

    Western blotting is a commonly used protein assay that combines the selectivity of electrophoretic separation and immunoassay. The technique is limited by long time, manual operation with mediocre reproducibility, and large sample consumption, typically 10-20 μg per assay. Western blots are also usually used to measure only one protein per assay with an additional housekeeping protein for normalization. Measurement of multiple proteins is possible; however, it requires stripping membranes of antibody and then reprobing with a second antibody. Miniaturized alternatives to Western blot based on microfluidic or capillary electrophoresis have been developed that enable higher-throughput, automation, and greater mass sensitivity. In one approach, proteins are separated by electrophoresis on a microchip that is dragged along a polyvinylidene fluoride membrane so that as proteins exit the chip they are captured on the membrane for immunoassay. In this work, we improve this method to allow multiplexed protein detection. Multiple injections made from the same sample can be deposited in separate tracks so that each is probed with a different antibody. To further enhance multiplexing capability, the electrophoresis channel dimensions were optimized for resolution while keeping separation and blotting times to less than 8 min. Using a 15 μm deep × 50 μm wide × 8.6 cm long channel, it is possible to achieve baseline resolution of proteins that differ by 5% in molecular weight, e.g., ERK1 (44 kDa) from ERK2 (42 kDa). This resolution allows similar proteins detected by cross-reactive antibodies in a single track. We demonstrate detection of 11 proteins from 9 injections from a single Jurkat cell lysate sample consisting of 400 ng of total protein using this procedure. Thus, multiplexed Western blots are possible without cumbersome stripping and reprobing steps. PMID:27270033

  15. Modulating the Growth and Imatinib Sensitivity of Chronic Myeloid Leukemia Stem/Progenitor Cells with Pullulan/MicroRNA Nanoparticles In Vitro.

    PubMed

    Ma, Wenjuan; Liu, Jian; Xie, Jundan; Zhang, Xiuyan; Zhou, Haixia; Yao, Hong; Zhang, Weiqi; Guo, Dawei; Zhu, Lingying; Xiao, Lun; Wu, Depei; Xu, Haiyan; Chen, Suning; Zhao, Yun

    2015-11-01

    Chronic myeloid leukemia (CML) originates from normal hematopoietic stem cells acquiring Philadelphia chromosome (Ph) to generate BCR-ABL fusion gene whose protein product has deregulated tyrosine kinase activity. Specific inhibitors against BCR-ABL, such as Imatinib mesylate (IM), have greatly improved CML management; however, no single agent is a cure yet. Delivery of microRNA (miRNA) using non-viral vectors has been utilized to inhibit various cancer cells; however, the efficacy of this approach to target CML stem/progenitor cells has not been elucidated. In this study, we firstly validated that spermine-introduced pullulan (Ps) was a robust non-viral vector for delivery of miRNA to CML cells, including the CD34+ cells from clinical isolates. We then found that the miR-181a/RALA (V-ral simian leukemia viral oncogene homolog A) axis was aberrantly expressed in the CML CD34+ cells. The delivery of miR-181a specifically inhibited the growth of CML CD34+ cells, possibly via the inhibition of RALA. In contrast, miR-181a did not evidently affect the normal hematopoietic CD34+ cells. In addition, miR-181a increased IM sensitivity of the CD34+ CML cells. Taken together, we have therefore demonstrated that the delivery of miR-181a using Ps to CML stem/progenitor cells leads to their growth inhibition and enhancement of IM sensitivity, which will possibly be beneficial to CML treatment. PMID:26554155

  16. Thermally multiplexed polymerase chain reaction.

    PubMed

    Phaneuf, Christopher R; Pak, Nikita; Saunders, D Curtis; Holst, Gregory L; Birjiniuk, Joav; Nagpal, Nikita; Culpepper, Stephen; Popler, Emily; Shane, Andi L; Jerris, Robert; Forest, Craig R

    2015-07-01

    Amplification of multiple unique genetic targets using the polymerase chain reaction (PCR) is commonly required in molecular biology laboratories. Such reactions are typically performed either serially or by multiplex PCR. Serial reactions are time consuming, and multiplex PCR, while powerful and widely used, can be prone to amplification bias, PCR drift, and primer-primer interactions. We present a new thermocycling method, termed thermal multiplexing, in which a single heat source is uniformly distributed and selectively modulated for independent temperature control of an array of PCR reactions. Thermal multiplexing allows amplification of multiple targets simultaneously-each reaction segregated and performed at optimal conditions. We demonstrate the method using a microfluidic system consisting of an infrared laser thermocycler, a polymer microchip featuring 1 μl, oil-encapsulated reactions, and closed-loop pulse-width modulation control. Heat transfer modeling is used to characterize thermal performance limitations of the system. We validate the model and perform two reactions simultaneously with widely varying annealing temperatures (48 °C and 68 °C), demonstrating excellent amplification. In addition, to demonstrate microfluidic infrared PCR using clinical specimens, we successfully amplified and detected both influenza A and B from human nasopharyngeal swabs. Thermal multiplexing is scalable and applicable to challenges such as pathogen detection where patients presenting non-specific symptoms need to be efficiently screened across a viral or bacterial panel. PMID:26339317

  17. Bond Percolation on Multiplex Networks

    NASA Astrophysics Data System (ADS)

    Hackett, A.; Cellai, D.; Gómez, S.; Arenas, A.; Gleeson, J. P.

    2016-04-01

    We present an analytical approach for bond percolation on multiplex networks and use it to determine the expected size of the giant connected component and the value of the critical bond occupation probability in these networks. We advocate the relevance of these tools to the modeling of multilayer robustness and contribute to the debate on whether any benefit is to be yielded from studying a full multiplex structure as opposed to its monoplex projection, especially in the seemingly irrelevant case of a bond occupation probability that does not depend on the layer. Although we find that in many cases the predictions of our theory for multiplex networks coincide with previously derived results for monoplex networks, we also uncover the remarkable result that for a certain class of multiplex networks, well described by our theory, new critical phenomena occur as multiple percolation phase transitions are present. We provide an instance of this phenomenon in a multiplex network constructed from London rail and European air transportation data sets.

  18. Thermally multiplexed polymerase chain reaction

    PubMed Central

    Phaneuf, Christopher R.; Pak, Nikita; Saunders, D. Curtis; Holst, Gregory L.; Birjiniuk, Joav; Nagpal, Nikita; Culpepper, Stephen; Popler, Emily; Shane, Andi L.; Jerris, Robert; Forest, Craig R.

    2015-01-01

    Amplification of multiple unique genetic targets using the polymerase chain reaction (PCR) is commonly required in molecular biology laboratories. Such reactions are typically performed either serially or by multiplex PCR. Serial reactions are time consuming, and multiplex PCR, while powerful and widely used, can be prone to amplification bias, PCR drift, and primer-primer interactions. We present a new thermocycling method, termed thermal multiplexing, in which a single heat source is uniformly distributed and selectively modulated for independent temperature control of an array of PCR reactions. Thermal multiplexing allows amplification of multiple targets simultaneously—each reaction segregated and performed at optimal conditions. We demonstrate the method using a microfluidic system consisting of an infrared laser thermocycler, a polymer microchip featuring 1 μl, oil-encapsulated reactions, and closed-loop pulse-width modulation control. Heat transfer modeling is used to characterize thermal performance limitations of the system. We validate the model and perform two reactions simultaneously with widely varying annealing temperatures (48 °C and 68 °C), demonstrating excellent amplification. In addition, to demonstrate microfluidic infrared PCR using clinical specimens, we successfully amplified and detected both influenza A and B from human nasopharyngeal swabs. Thermal multiplexing is scalable and applicable to challenges such as pathogen detection where patients presenting non-specific symptoms need to be efficiently screened across a viral or bacterial panel. PMID:26339317

  19. Structural measures for multiplex networks

    NASA Astrophysics Data System (ADS)

    Battiston, Federico; Nicosia, Vincenzo; Latora, Vito

    2014-03-01

    Many real-world complex systems consist of a set of elementary units connected by relationships of different kinds. All such systems are better described in terms of multiplex networks, where the links at each layer represent a different type of interaction between the same set of nodes rather than in terms of (single-layer) networks. In this paper we present a general framework to describe and study multiplex networks, whose links are either unweighted or weighted. In particular, we propose a series of measures to characterize the multiplexicity of the systems in terms of (i) basic node and link properties such as the node degree, and the edge overlap and reinforcement, (ii) local properties such as the clustering coefficient and the transitivity, and (iii) global properties related to the navigability of the multiplex across the different layers. The measures we introduce are validated on a genuinely multiplex data set of Indonesian terrorists, where information among 78 individuals are recorded with respect to mutual trust, common operations, exchanged communications, and business relationships.

  20. Structural measures for multiplex networks.

    PubMed

    Battiston, Federico; Nicosia, Vincenzo; Latora, Vito

    2014-03-01

    Many real-world complex systems consist of a set of elementary units connected by relationships of different kinds. All such systems are better described in terms of multiplex networks, where the links at each layer represent a different type of interaction between the same set of nodes rather than in terms of (single-layer) networks. In this paper we present a general framework to describe and study multiplex networks, whose links are either unweighted or weighted. In particular, we propose a series of measures to characterize the multiplexicity of the systems in terms of (i) basic node and link properties such as the node degree, and the edge overlap and reinforcement, (ii) local properties such as the clustering coefficient and the transitivity, and (iii) global properties related to the navigability of the multiplex across the different layers. The measures we introduce are validated on a genuinely multiplex data set of Indonesian terrorists, where information among 78 individuals are recorded with respect to mutual trust, common operations, exchanged communications, and business relationships.

  1. Advances in multiplex PCR: balancing primer efficiencies and improving detection success

    PubMed Central

    Sint, Daniela; Raso, Lorna; Traugott, Michael

    2012-01-01

    1. Multiplex PCR is a valuable tool in many biological studies but it is a multifaceted procedure that has to be planned and optimised thoroughly to achieve robust and meaningful results. In particular, primer concentrations have to be adjusted to assure an even amplification of all targeted DNA fragments. Until now, total DNA extracts were used for balancing primer efficiencies; however, the applicability for comparisons between taxa or different multiple-copy genes was limited owing to the unknown number of template molecules present per total DNA. 2. Based on a multiplex system developed to track trophic interactions in high Alpine arthropods, we demonstrate a fast and easy way of generating standardised DNA templates. These were then used to balance the amplification success for the different targets and to subsequently determine the sensitivity of each primer pair in the multiplex PCR. 3. In the current multiplex assay, this approach led to an even amplification success for all seven targeted DNA fragments. Using this balanced multiplex PCR, methodological bias owing to variation in primer efficiency will be avoided when analysing field-derived samples. 4. The approach outlined here allows comparing multiplex PCR sensitivity, independent of the investigated species, genome size or the targeted genes. The application of standardised DNA templates not only makes it possible to optimise primer efficiency within a given multiplex PCR, but it also offers to adjust and/or to compare the sensitivity between different assays. Along with other factors that influence the success of multiplex reactions, and which we discuss here in relation to the presented detection system, the adoption of this approach will allow for direct comparison of multiplex PCR data between systems and studies, enhancing the utility of this assay type. PMID:23549328

  2. High-throughput multiplexed xMAP Luminex array panel for detection of twenty two medically important mosquito-borne arboviruses based on innovations in synthetic biology.

    PubMed

    Glushakova, Lyudmyla G; Bradley, Andrea; Bradley, Kevin M; Alto, Barry W; Hoshika, Shuichi; Hutter, Daniel; Sharma, Nidhi; Yang, Zunyi; Kim, Myong-Jung; Benner, Steven A

    2015-03-01

    Mosquito-borne arboviruses are emerging world-wide as important human and animal pathogens. This makes assays for their accurate and rapid identification essential for public health, epidemiological, ecological studies. Over the past decade, many mono- and multiplexed assays targeting arboviruses nucleic acids have been reported. None has become established for the routine identification of multiple viruses in a "single tube" setting. With increasing multiplexing, the detection of viral RNAs is complicated by noise, false positives and negatives. In this study, an assay was developed that avoids these problems by combining two new kinds of nucleic acids emerging from the field of synthetic biology. The first is a "self-avoiding molecular recognition system" (SAMRS), which enables high levels of multiplexing. The second is an "artificially expanded genetic information system" (AEGIS), which enables clean PCR amplification in nested PCR formats. A conversion technology was used to place AEGIS component into amplicon, improving their efficiency of hybridization on Luminex beads. When Luminex "liquid microarrays" are exploited for downstream detection, this combination supports single-tube PCR amplification assays that can identify 22 mosquito-borne RNA viruses from the genera Flavivirus, Alphavirus, Orthobunyavirus. The assay differentiates between closely-related viruses, as dengue, West Nile, Japanese encephalitis, and the California serological group. The performance and the sensitivity of the assay were evaluated with dengue viruses and infected mosquitoes; as few as 6-10 dengue virions can be detected in a single mosquito.

  3. High-throughput Multiplexed xMAP Luminex Array Panel for Detection of Twenty TWO Medically Important Mosquito-borne Arboviruses based on Innovations in Synthetic Biology

    PubMed Central

    Glushakova, Lyudmyla G.; Bradley, Andrea; Bradley, Kevin M.; Alto, Barry W.; Hoshika, Shuichi; Hutter, Daniel; Sharma, Nidhi; Yang, Zunyi; Kim, Myong-Jung; Benner, Steven A.

    2015-01-01

    Mosquito-borne arboviruses are emerging world-wide as important human and animal pathogens. This makes assays for their accurate and rapid identification essential for public health, epidemiological, ecological studies. Over the past decade, many mono- and multiplexed assays targeting arboviruses nucleic acids have been reported. None has become established for the routine identification of multiple viruses in a “single tube” setting. With increasing multiplexing, the detection of viral RNAs is complicated by noise, false positives and negatives. In this study, an assay was developed that avoids these problems by combining two new kinds of nucleic acids emerging from the field of synthetic biology. The first is a “self-avoiding molecular recognition system” (SAMRS), which enables high levels of multiplexing. The second is an “artificially expanded genetic information system” (AEGIS), which enables clean PCR amplification in nested PCR formats. A conversion technology was used to place AEGIS component into amplicon, improving their efficiency of hybridization on Luminex beads. When Luminex “liquid microarrays” are exploited for downstream detection, this combination supports single-tube PCR amplification assays that can identify 22 mosquito-borne RNA viruses from the genera Flavivirus, Alphavirus, Orthobunyavirus. The assay differentiates between closely-related viruses, as dengue, West Nile, Japanese encephalitis, and the California serological group. The performance and the sensitivity of the assay were evaluated with dengue viruses and infected mosquitoes; as few as 6–10 dengue virions can be detected in a single mosquito. PMID:25680538

  4. Multiplex PCR assays for the detection of Vibrio alginolyticus, Vibrio parahaemolyticus, Vibrio vulnificus, and Vibrio cholerae with an internal amplification control.

    PubMed

    Wei, Shuang; Zhao, Hui; Xian, Yuyin; Hussain, Malik A; Wu, Xiyang

    2014-06-01

    A multiplex polymerase chain reaction (PCR) assay that can simultaneously detect 4 major Vibrio spp., Vibrio alginolyticus, Vibrio parahaemolyticus, Vibrio vulnificus, and Vibrio cholerae, in the presence of an internal amplification control (IAC) was developed. Species-specific PCR primers were designed based on the gyrB gene for V. alginolyticus, the collagenase gene for V. parahaemolyticus, the vvhA gene for V. vulnificus, and the ompW gene for V. cholerae. Additionally, an IAC primer pair was designed in conserved regions of the bacterial 16S rRNA gene that is used to indicate false-negative results. A multiplex PCR method was developed after optimization of the reaction conditions. The specificity of the PCR was validated by using 83 Vibrio strains and 10 other non-Vibrio bacterial species. The detection limit of the PCR was 10 CFU per tube for V. alginolyticus, V. parahaemolyticus, V. vulnificus, and 10(5) CFU per tube for V. cholerae in mixed conditions. This method was used to identify 69 suspicious Vibrio isolates, and the results were consistent with physiological and biochemical tests. This multiplex PCR method proved to be rapid, sensitive, and specific. The existence of IAC could successfully eliminate false-negative results for the detection of V. alginolyticus, V. parahaemolyticus, V. vulnificus, and V. cholerae.

  5. Helicity multiplexed broadband metasurface holograms

    PubMed Central

    Wen, Dandan; Yue, Fuyong; Li, Guixin; Zheng, Guoxing; Chan, Kinlong; Chen, Shumei; Chen, Ming; Li, King Fai; Wong, Polis Wing Han; Cheah, Kok Wai; Yue Bun Pun, Edwin; Zhang, Shuang; Chen, Xianzhong

    2015-01-01

    Metasurfaces are engineered interfaces that contain a thin layer of plasmonic or dielectric nanostructures capable of manipulating light in a desirable manner. Advances in metasurfaces have led to various practical applications ranging from lensing to holography. Metasurface holograms that can be switched by the polarization state of incident light have been demonstrated for achieving polarization multiplexed functionalities. However, practical application of these devices has been limited by their capability for achieving high efficiency and high image quality. Here we experimentally demonstrate a helicity multiplexed metasurface hologram with high efficiency and good image fidelity over a broad range of frequencies. The metasurface hologram features the combination of two sets of hologram patterns operating with opposite incident helicities. Two symmetrically distributed off-axis images are interchangeable by controlling the helicity of the input light. The demonstrated helicity multiplexed metasurface hologram with its high performance opens avenues for future applications with functionality switchable optical devices. PMID:26354497

  6. Liquid Hybridization and Solid Phase Detection: A Highly Sensitive and Accurate Strategy for MicroRNA Detection in Plants and Animals.

    PubMed

    Li, Fosheng; Mei, Lanju; Zhan, Cheng; Mao, Qiang; Yao, Min; Wang, Shenghua; Tang, Lin; Chen, Fang

    2016-01-01

    MicroRNAs (miRNAs) play important roles in nearly every aspect of biology, including physiological, biochemical, developmental and pathological processes. Therefore, a highly sensitive and accurate method of detection of miRNAs has great potential in research on theory and application, such as the clinical approach to medicine, animal and plant production, as well as stress response. Here, we report a strategic method to detect miRNAs from multicellular organisms, which mainly includes liquid hybridization and solid phase detection (LHSPD); it has been verified in various species and is much more sensitive than traditional biotin-labeled Northern blots. By using this strategy and chemiluminescent detection with digoxigenin (DIG)-labeled or biotin-labeled oligonucleotide probes, as low as 0.01-0.25 fmol [for DIG-CDP Star (disodium2-chloro-5-(4-methoxyspiro{1,2-dioxetane-3,2'-(5'-chloro)tricyclo[3.3.1.13,7]decan}-4-yl)phenyl phosphate) system], 0.005-0.1 fmol (for biotin-CDP Star system), or 0.05-0.5 fmol (for biotin-luminol system) of miRNA can be detected and one-base difference can be distinguished between miRNA sequences. Moreover, LHSPD performed very well in the quantitative analysis of miRNAs, and the whole process can be completed within about 9 h. The strategy of LHSPD provides an effective solution for rapid, accurate, and sensitive detection and quantitative analysis of miRNAs in plants and animals. PMID:27598139

  7. Liquid Hybridization and Solid Phase Detection: A Highly Sensitive and Accurate Strategy for MicroRNA Detection in Plants and Animals

    PubMed Central

    Li, Fosheng; Mei, Lanju; Zhan, Cheng; Mao, Qiang; Yao, Min; Wang, Shenghua; Tang, Lin; Chen, Fang

    2016-01-01

    MicroRNAs (miRNAs) play important roles in nearly every aspect of biology, including physiological, biochemical, developmental and pathological processes. Therefore, a highly sensitive and accurate method of detection of miRNAs has great potential in research on theory and application, such as the clinical approach to medicine, animal and plant production, as well as stress response. Here, we report a strategic method to detect miRNAs from multicellular organisms, which mainly includes liquid hybridization and solid phase detection (LHSPD); it has been verified in various species and is much more sensitive than traditional biotin-labeled Northern blots. By using this strategy and chemiluminescent detection with digoxigenin (DIG)-labeled or biotin-labeled oligonucleotide probes, as low as 0.01–0.25 fmol [for DIG-CDP Star (disodium2-chloro-5-(4-methoxyspiro{1,2-dioxetane-3,2′-(5′-chloro)tricyclo[3.3.1.13,7]decan}-4-yl)phenyl phosphate) system], 0.005–0.1 fmol (for biotin-CDP Star system), or 0.05–0.5 fmol (for biotin-luminol system) of miRNA can be detected and one-base difference can be distinguished between miRNA sequences. Moreover, LHSPD performed very well in the quantitative analysis of miRNAs, and the whole process can be completed within about 9 h. The strategy of LHSPD provides an effective solution for rapid, accurate, and sensitive detection and quantitative analysis of miRNAs in plants and animals. PMID:27598139

  8. Integrated multiplexed capillary electrophoresis system

    DOEpatents

    Yeung, Edward S.; Tan, Hongdong

    2002-05-14

    The present invention provides an integrated multiplexed capillary electrophoresis system for the analysis of sample analytes. The system integrates and automates multiple components, such as chromatographic columns and separation capillaries, and further provides a detector for the detection of analytes eluting from the separation capillaries. The system employs multiplexed freeze/thaw valves to manage fluid flow and sample movement. The system is computer controlled and is capable of processing samples through reaction, purification, denaturation, pre-concentration, injection, separation and detection in parallel fashion. Methods employing the system of the invention are also provided.

  9. Optical Multiplex Systems For Vehicles

    NASA Astrophysics Data System (ADS)

    Rogers, Wesley A.

    1980-09-01

    Optical multiplex technology, presently in vogue in many segments of industry, is now under scrutiny at General Motors. We are evaluating this technology as a means of simplifying the vehicle harness, reducing weight, eliminating electromagnetic interference, and providing drastically new interior styling options. Door, seat, steering column, forward and rear harness vehicle assemblies, are replaced with a single fiber optic cable in each area. A four bit microprocessor at the top of the steering column, and in each door, multiplexes control console button commands over fiber optic cables. A microprocessor at the other end of the cables decodes the optical signals and operates seats, windows, windshield wipers, etc.

  10. Turing patterns in multiplex networks

    NASA Astrophysics Data System (ADS)

    Asllani, Malbor; Busiello, Daniel M.; Carletti, Timoteo; Fanelli, Duccio; Planchon, Gwendoline

    2014-10-01

    The theory of patterns formation for a reaction-diffusion system defined on a multiplex is developed by means of a perturbative approach. The interlayer diffusion constants act as a small parameter in the expansion and the unperturbed state coincides with the limiting setting where the multiplex layers are decoupled. The interaction between adjacent layers can seed the instability of a homogeneous fixed point, yielding self-organized patterns which are instead impeded in the limit of decoupled layers. Patterns on individual layers can also fade away due to cross-talking between layers. Analytical results are compared to direct simulations.

  11. Short-interfering RNA-mediated silencing of thioredoxin reductase 1 alters the sensitivity of HeLa cells toward cadmium.

    PubMed

    Nishimoto, Michie; Sakaue, Motoharu; Hara, Shuntaro

    2006-03-01

    The mammalian thioredoxin reductase (TrxR) is a selenocysteine-containing flavoprotein that regulates the thioredoxin system, one of the major systems that maintain the intracellular redox balance. We previously reported that cytosolic TrxR (TrxR1), one of three mammalian TrxR isozymes, was induced by treatment with cadmium. In the present study, to study the role of cadmium-induced TrxR1 in cellular defense, we silenced the expression of TrxR1 in HeLa cells by using small interfering RNA and examined the effect of TrxR1 silencing on the sensitivity of the cells toward cadmium. We found that the gene silencing of TrxR1 had a dual effect on cadmium-induced cell death, depending on the concentration of cadmium. The TrxR1 silencing increased the sensitivity toward a low dose (less than 10 microM) of cadmium but decreased the sensitivity toward a high dose of cadmium. These results suggested that TrxR1 might play an important role in the cellular defense system against cadmium in two ways. TrxR1 might rescue the cells from a low dose of cadmium-induced moderate injury, while it might promote the death of cells severely injured by a high dose of cadmium.

  12. Multiplexed label-free optical biosensor for medical diagnostics.

    PubMed

    Bottazzi, Barbara; Fornasari, Lucia; Frangolho, Ana; Giudicatti, Silvia; Mantovani, Alberto; Marabelli, Franco; Marchesini, Gerardo; Pellacani, Paola; Therisod, Rita; Valsesia, Andrea

    2014-01-01

    This paper describes a new multiplexed label-free biosensor. The detection technology is based on nanostructured gold-polymer surfaces. These surfaces support surface plasmon resonance modes that can be probed by a miniaturized optical setup. The optical characterization of the sensing chip shows the sensitivity and the limit-of-detection to refractive index changes. Moreover, by studying the progressive adhesion of molecular monolayers of polyelectrolytes, the decay of the plasmonic mode electric field above the surface has been reconstructed. A multiplexed label-free biosensing device is then described and characterized in terms of sensitivity, lateral resolution, and sensitivity to a model biological assay. The sensitivity in imaging mode of the device is of the order of 10-6 refractive index units, while the measured lateral resolution is 6.25 μm within a field of view of several tenths of mm2, making the instrument unique in terms of multiplexing capability. Finally, the proof-of-concept application of the technology as a point-of-care diagnostic tool for an inflammatory marker is demonstrated. PMID:24474511

  13. A Rapid and Sensitive Method to Identify and Differentiate Salmonella enterica Serotype Typhimurium and Salmonella enterica Serotype 4,[5],12:i:- by Combining Traditional Serotyping and Multiplex Polymerase Chain Reaction

    PubMed Central

    Barco, Lisa; Lettini, Antonia Anna; Ramon, Elena; Longo, Alessandra; Saccardin, Cristina; Pozza, Maria Cristina Dalla

    2011-01-01

    Abstract Salmonella enterica subspecies enterica serotype 4,[5],12:i:- is an emerging serovar considered as a monophasic variant of Salmonella enterica serotype Typhimurium. The antigenic and genetic similarity between Salmonella 4,[5],12:i:- and Salmonella Typhimurium suggests that they may behave in a similar way and represent a comparable threat to public health. As serotyping alone does not necessarily provide for identification of Salmonella 4,[5],12:i:- and its differentiation from Salmonella Typhimurium, a method that combines traditional serotyping and a multiplex polymerase chain reaction has been tested on 208 strains serotyped as Salmonella 4,[5],12:i:-, Salmonella Typhimurium, and similar serovars of serogroup B sharing the same phase-1 antigen “i.” For 191 strains, the combined method fully confirmed the results provided by traditional serotyping, whereas for 17 strains of Salmonella 4,[5],12:i:- and Salmonella Typhimurium some inconsistencies emerged between the two methods. The combined method resulted in a more accurate and faster identification of these two relevant serovars. PMID:21247297

  14. MicroRNA-137 inhibits tumor growth and sensitizes chemosensitivity to paclitaxel and cisplatin in lung cancer

    PubMed Central

    Ge, Xin; Jiang, Cheng-Fei; Shi, Zhu-Mei; Li, Dong-Mei; Liu, Wei-Tao; Yu, Xiaobo; Shu, Yong-Qian

    2016-01-01

    Chemotherapy resistance frequently drives tumour progression. However, the underlying molecular mechanisms are poorly characterized. In this study, we explored miR-137's role in the chemosensitivity of lung cancer. We found that the expression level of miR-137 is down-regulated in the human lung cancer tissues and the resistant cells strains: A549/paclitaxel(A549/PTX) and A549/cisplatin (A549/CDDP) when compared with lung cancer A549 cells. Moreover, we found that overe-expression of miR-137 inhibited cell proliferation, migration, cell survival and arrest the cell cycle in G1 phase in A549/PTX and A549/CDDP. Furthermore, Repression of miR-137 significantly promoted cell growth, migration, cell survival and cell cycle G1/S transition in A549 cells. We further demonstrated that the tumor suppressive role of miR-137 was mediated by negatively regulating Nuclear casein kinase and cyclin-dependent kinase substrate1(NUCKS1) protein expression. Importantly, miR-137 inhibits A549/PTX, A549/CDDP growth and angiogenesis in vivo. Our study is the first to identify the tumor suppressive role of over-expressed miR-137 in chemosensitivity. Identification of a novel miRNA-mediated pathway that regulates chemosensitivity in lung cancer will facilitate the development of novel therapeutic strategies in the future. PMID:26989074

  15. A LexA-related protein regulates redox-sensitive expression of the cyanobacterial RNA helicase, crhR

    PubMed Central

    Patterson-Fortin, Laura M.; Colvin, Kimberley R.; Owttrim, George W.

    2006-01-01

    Expression of the cyanobacterial DEAD-box RNA helicase, crhR, is regulated in response to conditions, which elicit reduction of the photosynthetic electron transport chain. A combination of electrophoretic mobility shift assay (EMSA), DNA affinity chromatography and mass spectrometry identified that a LexA-related protein binds specifically to the crhR gene. Transcript analysis indicates that lexA and crhR are divergently expressed, with lexA and crhR transcripts accumulating differentially under conditions, which respectively oxidize and reduce the electron transport chain. In addition, expression of the Synechocystis lexA gene is not DNA damage inducible and its amino acid sequence lacks two of three residues required for activity of prototypical LexA proteins, which repress expression of DNA repair genes in a range of prokaryotes. A direct effect of recombinant LexA protein on crhR expression was confirmed from the observation that LexA reduces crhR expression in a linear manner in an in vitro transcription/translation assay. The results indicate that the Synechocystis LexA-related protein functions as a regulator of redox-responsive crhR gene expression, and not DNA damage repair genes. PMID:16840531

  16. Multiplex SYBR Green Real-Time PCR Assay for Detection of Respiratory Viruses

    PubMed Central

    Sultani, Mozhdeh; Mokhtari Azad, Talat; Eshragian, Mohammadreza; Shadab, Azadeh; Naseri, Maryam; Eilami, Owrang; Yavarian, Jila

    2015-01-01

    Background: It is often difficult for a physician to distinguish between viral and bacterial causes of respiratory infections and this may result in overuse of antibiotics. In many cases of community-acquired respiratory infections, clinicians treat patients empirically. The development of molecular methods for direct detection of viruses has been progressed recently. Objectives: The objective of this study was recognizing the panel of respiratory RNA viruses by multiplex SYBR Green real-time polymerase chain reaction (PCR). Materials and Methods: Randomized 172 influenza-negative respiratory specimens of all age groups of hospitalized patients were collected. After RNA extraction, cDNA was synthesized. Three SYBR Green multiplex real-time PCR assays were developed for simultaneous detection of 12 respiratory RNA viruses. Each set of multiplex methods detected four viruses, the first set: respiratory syncytial virus, human metapneumovirus, rhinovirus, enterovirus; the second set: parainfluenza viruses 1 - 4 (PIV1-4); the third set: coronaviruses NL63, 229E, severe acute respiratory syndrome (SARS), and OC43. Results: Application of the multiplex SYBR Green real-time PCR in clinical samples from 172 patients in a one-year study resulted in detection of 19 (11.04%) PIV3, 9 (5.23%) PIV4, and 1 (0.58%) coronavirus NL63. All the positive samples were detected during December to March (2011 - 2012). Conclusions: Multiplex SYBR Green real-time PCR is a rapid and relatively inexpensive method for detection of respiratory viruses. PMID:26468358

  17. Point-of-care multiplexed assays of nucleic acids using microcapillary-based loop-mediated isothermal amplification.

    PubMed

    Zhang, Yi; Zhang, Lu; Sun, Jiashu; Liu, Yulei; Ma, Xingjie; Cui, Shangjin; Ma, Liying; Xi, Jianzhong Jeff; Jiang, Xingyu

    2014-07-15

    This report demonstrates a straightforward, robust, multiplexed and point-of-care microcapillary-based loop-mediated isothermal amplification (cLAMP) for assaying nucleic acids. This assay integrates capillaries (glass or plastic) to introduce and house sample/reagents, segments of water droplets to prevent contamination, pocket warmers to provide heat, and a hand-held flashlight for a visual readout of the fluorescent signal. The cLAMP system allows the simultaneous detection of two RNA targets of human immunodeficiency virus (HIV) from multiple plasma samples, and achieves a high sensitivity of two copies of standard plasmid. As few nucleic acid detection methods can be wholly independent of external power supply and equipment, our cLAMP holds great promise for point-of-care applications in resource-poor settings. PMID:24937125

  18. Development of a multiplex real-time RT-PCR assay for simultaneous detection of dengue and chikungunya viruses.

    PubMed

    Cecilia, D; Kakade, M; Alagarasu, K; Patil, J; Salunke, A; Parashar, D; Shah, P S

    2015-01-01

    Dengue and chikungunya viruses co-circulate and cause infections that start with similar symptoms but progress to radically different outcomes. Therefore, an early diagnostic test that can differentiate between the two is needed. A single-step multiplex real-time RT-PCR assay was developed that can simultaneously detect and quantitate RNA of all dengue virus (DENV) serotypes and chikungunya virus (CHIKV). The sensitivity was 100 % for DENV and 95.8 % for CHIKV, whilst the specificity was 100 % for both viruses when compared with conventional RT-PCR. The detection limit ranged from 1 to 50 plaque-forming units. The assay was successfully used for differential diagnosis of dengue and chikungunya in Pune, where the viruses co-circulate.

  19. Multiplexed Recombinase Polymerase Amplification Assay To Detect Intestinal Protozoa.

    PubMed

    Crannell, Zachary; Castellanos-Gonzalez, Alejandro; Nair, Gayatri; Mejia, Rojelio; White, A Clinton; Richards-Kortum, Rebecca

    2016-02-01

    This work describes a proof-of-concept multiplex recombinase polymerase amplification (RPA) assay with lateral flow readout that is capable of simultaneously detecting and differentiating DNA from any of the diarrhea-causing protozoa Giardia, Cryptosporidium, and Entamoeba. Together, these parasites contribute significantly to the global burden of diarrheal illness. Differential diagnosis of these parasites is traditionally accomplished via stool microscopy. However, microscopy is insensitive and can miss up to half of all cases. DNA-based diagnostics such as polymerase chain reaction (PCR) are far more sensitive; however, they rely on expensive thermal cycling equipment, limiting their availability to centralized reference laboratories. Isothermal DNA amplification platforms, such as the RPA platform used in this study, alleviate the need for thermal cycling equipment and have the potential to broaden access to more sensitive diagnostics. Until now, multiplex RPA assays have not been developed that are capable of simultaneously detecting and differentiating infections caused by different pathogens. We developed a multiplex RPA assay to detect the presence of DNA from Giardia, Cryptosporidium, and Entamoeba. The multiplex assay was characterized using synthetic DNA, where the limits-of-detection were calculated to be 403, 425, and 368 gene copies per reaction of the synthetic Giardia, Cryptosporidium, and Entamoeba targets, respectively (roughly 1.5 orders of magnitude higher than for the same targets in a singleplex RPA assay). The multiplex assay was also characterized using DNA extracted from live parasites spiked into stool samples where the limits-of-detection were calculated to be 444, 6, and 9 parasites per reaction for Giardia, Cryptosporidium, and Entamoeba parasites, respectively. This proof-of-concept assay may be reconfigured to detect a wide variety of targets by re-designing the primer and probe sequences.

  20. Multiplexed Recombinase Polymerase Amplification Assay To Detect Intestinal Protozoa.

    PubMed

    Crannell, Zachary; Castellanos-Gonzalez, Alejandro; Nair, Gayatri; Mejia, Rojelio; White, A Clinton; Richards-Kortum, Rebecca

    2016-02-01

    This work describes a proof-of-concept multiplex recombinase polymerase amplification (RPA) assay with lateral flow readout that is capable of simultaneously detecting and differentiating DNA from any of the diarrhea-causing protozoa Giardia, Cryptosporidium, and Entamoeba. Together, these parasites contribute significantly to the global burden of diarrheal illness. Differential diagnosis of these parasites is traditionally accomplished via stool microscopy. However, microscopy is insensitive and can miss up to half of all cases. DNA-based diagnostics such as polymerase chain reaction (PCR) are far more sensitive; however, they rely on expensive thermal cycling equipment, limiting their availability to centralized reference laboratories. Isothermal DNA amplification platforms, such as the RPA platform used in this study, alleviate the need for thermal cycling equipment and have the potential to broaden access to more sensitive diagnostics. Until now, multiplex RPA assays have not been developed that are capable of simultaneously detecting and differentiating infections caused by different pathogens. We developed a multiplex RPA assay to detect the presence of DNA from Giardia, Cryptosporidium, and Entamoeba. The multiplex assay was characterized using synthetic DNA, where the limits-of-detection were calculated to be 403, 425, and 368 gene copies per reaction of the synthetic Giardia, Cryptosporidium, and Entamoeba targets, respectively (roughly 1.5 orders of magnitude higher than for the same targets in a singleplex RPA assay). The multiplex assay was also characterized using DNA extracted from live parasites spiked into stool samples where the limits-of-detection were calculated to be 444, 6, and 9 parasites per reaction for Giardia, Cryptosporidium, and Entamoeba parasites, respectively. This proof-of-concept assay may be reconfigured to detect a wide variety of targets by re-designing the primer and probe sequences. PMID:26669715

  1. Nuclear modifier MTO2 modulates the aminoglycoside-sensitivity of mitochondrial 15S rRNA C1477G mutation in Saccharomyces cerevisiae.

    PubMed

    He, Xiangyu; Zhu, Xiaoyu; Wang, Xuexiang; Wang, Wei; Dai, Yu; Yan, Qingfeng

    2013-01-01

    The phenotypic manifestations of mitochondrial DNA (mtDNA) mutations are modulated by mitochondrial DNA haplotypes, nuclear modifier genes and environmental factors. The yeast mitochondrial 15S rRNA C1477G (P(R) or P(R) 454) mutation corresponds to the human 12S rRNA C1494T and A1555G mutations, which are well known as primary factors for aminoglycoside-induced nonsyndromic deafness. Here we report that the deletion of the nuclear modifier gene MTO2 suppressed the aminoglycoside-sensitivity of mitochondrial 15S rRNA C1477G mutation in Saccharomyces cerevisiae. First, the strain with a single mtDNA C1477G mutation exhibited hypersensitivity to neomycin. Functional assays indicated that the steady-state transcription level of mitochondrial DNA, the mitochondrial respiratory rate, and the membrane potential decreased significantly after neomycin treatment. The impaired mitochondria could not produce sufficient energy to maintain cell viability. Second, when the mto2 null and the mitochondrial C1477G mutations co-existed (mto2(P(R))), the oxygen consumption rate in the double mutant decreased markedly compared to that of the control strains (MTO2(P(S)), mto2(P(S)) and MTO2(P(R))). The expression levels of the key glycolytic genes HXK2, PFK1 and PYK1 in the mto2(P(R)) strain were stimulated by neomycin and up-regulated by 89%, 112% and 55%, respectively. The enhanced glycolysis compensated for the respiratory energy deficits, and could be inhibited by the glycolytic enzyme inhibitor. Our findings in yeast will provide a new insight into the pathogenesis of human deafness.

  2. IsoDOT Detects Differential RNA-isoform Expression/Usage with respect to a Categorical or Continuous Covariate with High Sensitivity and Specificity

    PubMed Central

    Sun, Wei; Liu, Yufeng; Crowley, James J.; Chen, Ting-Hued; Zhou, Hua; Chu, Haitao; Huang, Shunping; Kuan, Pei-Fen; Li, Yuan; Miller, Darla R.; Shaw, Ginger D.; Wu, Yichao; Zhabotynsky, Vasyl; McMillan, Leonard; Zou, Fei; Sullivan, Patrick F.; de Villena, Fernando Pardo-Manuel

    2015-01-01

    We have developed a statistical method named IsoDOT to assess differential isoform expression (DIE) and differential isoform usage (DIU) using RNA-seq data. Here isoform usage refers to relative isoform expression given the total expression of the corresponding gene. IsoDOT performs two tasks that cannot be accomplished by existing methods: to test DIE/DIU with respect to a continuous covariate, and to test DIE/DIU for one case versus one control. The latter task is not an uncommon situation in practice, e.g., comparing the paternal and maternal alleles of one individual or comparing tumor and normal samples of one cancer patient. Simulation studies demonstrate the high sensitivity and specificity of IsoDOT. We apply IsoDOT to study the effects of haloperidol treatment on the mouse transcriptome and identify a group of genes whose isoform usages respond to haloperidol treatment. PMID:26617424

  3. 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

  4. Mechanistic study on the nuclear modifier gene MSS1 mutation suppressing neomycin sensitivity of the mitochondrial 15S rRNA C1477G mutation in Saccharomyces cerevisiae.

    PubMed

    Zhou, Qiyin; Wang, Wei; He, Xiangyu; Zhu, Xiaoyu; Shen, Yaoyao; Yu, Zhe; Wang, Xuexiang; Qi, Xuchen; Zhang, Xuan; Fan, Mingjie; Dai, Yu; Yang, Shuxu; Yan, Qingfeng

    2014-01-01

    The phenotypic manifestation of mitochondrial DNA (mtDNA) mutations can be modulated by nuclear genes and environmental factors. However, neither the interaction among these factors nor their underlying mechanisms are well understood. The yeast Saccharomyces cerevisiae mtDNA 15S rRNA C1477G mutation (PR) corresponds to the human 12S rRNA A1555G mutation. Here we report that a nuclear modifier gene mss1 mutation suppresses the neomycin-sensitivity phenotype of a yeast C1477G mutant in fermentable YPD medium. Functional assays show that the mitochondrial function of the yeast C1477G mutant was impaired severely in YPD medium with neomycin. Moreover, the mss1 mutation led to a significant increase in the steady-state level of HAP5 (heme activated protein), which greatly up-regulated the expression of glycolytic transcription factors RAP1, GCR1, and GCR2 and thus stimulated glycolysis. Furthermore, the high expression of the key glycolytic enzyme genes HXK2, PFK1 and PYK1 indicated that enhanced glycolysis not only compensated for the ATP reduction from oxidative phosphorylation (OXPHOS) in mitochondria, but also ensured the growth of the mss1(PR) mutant in YPD medium with neomycin. This study advances our understanding of the phenotypic manifestation of mtDNA mutations.

  5. Robert Feulgen Prize Lecture 1999. Detection and amplification systems for sensitive, multiple-target DNA and RNA in situ hybridization: looking inside cells with a spectrum of colors.

    PubMed

    Speel, E J

    1999-08-01

    In situ hybridization (ISH) is a powerful technique for localizing specific nucleic acid sequences (DNA, RNA) in microscopic preparations of tissues, cells, chromosomes, and linear DNA fibers. To date, a wide variety of research and diagnostic applications of ISH have been described, making the technique an integral part of studies concerning gene mapping, gene expression, RNA processing and transport, the three-dimensional organization of the nucleus, tumor genetics, microbial infections, and prenatal diagnosis. In this review, I first describe the ISH procedure in short and then focus on the currently available non-radioactive probe-labeling and cytochemical detection methodologies that are utilized to visualize one or multiple different nucleic acid targets in situ with different colors. Special emphasis is placed on the procedures applying fluorescence and brightfield microscopy, the simultaneous detection of nucleic acids and proteins by combined ISH and immunocytochemistry, and, in addition, on the recent progress that has been made with the introduction of signal amplification procedures to increase the detection sensitivity of ISH. Finally, a comparison of fluorescence, enzyme cytochemical, and colloidal gold silver probe detection systems will be presented, and possible future directions of in situ nucleic acid detection will be discussed.

  6. Mechanistic Study on the Nuclear Modifier Gene MSS1 Mutation Suppressing Neomycin Sensitivity of the Mitochondrial 15S rRNA C1477G Mutation in Saccharomyces cerevisiae

    PubMed Central

    Zhou, Qiyin; Wang, Wei; He, Xiangyu; Zhu, Xiaoyu; Shen, Yaoyao; Yu, Zhe; Wang, Xuexiang; Qi, Xuchen; Zhang, Xuan; Fan, Mingjie; Dai, Yu; Yang, Shuxu; Yan, Qingfeng

    2014-01-01

    The phenotypic manifestation of mitochondrial DNA (mtDNA) mutations can be modulated by nuclear genes and environmental factors. However, neither the interaction among these factors nor their underlying mechanisms are well understood. The yeast Saccharomyces cerevisiae mtDNA 15S rRNA C1477G mutation (PR) corresponds to the human 12S rRNA A1555G mutation. Here we report that a nuclear modifier gene mss1 mutation suppresses the neomycin-sensitivity phenotype of a yeast C1477G mutant in fermentable YPD medium. Functional assays show that the mitochondrial function of the yeast C1477G mutant was impaired severely in YPD medium with neomycin. Moreover, the mss1 mutation led to a significant increase in the steady-state level of HAP5 (heme activated protein), which greatly up-regulated the expression of glycolytic transcription factors RAP1, GCR1, and GCR2 and thus stimulated glycolysis. Furthermore, the high expression of the key glycolytic enzyme genes HXK2, PFK1 and PYK1 indicated that enhanced glycolysis not only compensated for the ATP reduction from oxidative phosphorylation (OXPHOS) in mitochondria, but also ensured the growth of the mss1(PR) mutant in YPD medium with neomycin. This study advances our understanding of the phenotypic manifestation of mtDNA mutations. PMID:24595024

  7. Bixin regulates mRNA expression involved in adipogenesis and enhances insulin sensitivity in 3T3-L1 adipocytes through PPAR{gamma} activation

    SciTech Connect

    Takahashi, Nobuyuki; Goto, Tsuyoshi; Taimatsu, Aki; Egawa, Kahori; Katoh, Sota; Kusudo, Tatsuya; Sakamoto, Tomoya; Ohyane, Chie; Lee, Joo-Young; Kim, Young-il; Uemura, Taku; Hirai, Shizuka; Kawada, Teruo

    2009-12-25

    Insulin resistance is partly due to suppression of insulin-induced glucose uptake into adipocytes. The uptake is dependent on adipocyte differentiation, which is controlled at mRNA transcription level. The peroxisome proliferator-activated receptor (PPAR), a ligand-regulated nuclear receptor, is involved in the differentiation. Many food-derived compounds serve as ligands to activate or inactivate PPAR. In this study, we demonstrated that bixin and norbixin (annatto extracts) activate PPAR{gamma} by luciferase reporter assay using GAL4-PPAR chimera proteins. To examine the effects of bixin on adipocytes, 3T3-L1 adipocytes were treated with bixin or norbixin. The treatment induced mRNA expression of PPAR{gamma} target genes such as adipocyte-specific fatty acid-binding protein (aP2), lipoprotein lipase (LPL), and adiponectin in differentiated 3T3-L1 adipocytes and enhanced insulin-dependent glucose uptake. The observations indicate that bixin acts as an agonist of PPAR{gamma} and enhances insulin sensitivity in 3T3-L1 adipocytes, suggesting that bixin is a valuable food-derived compound as a PPAR ligand to regulate lipid metabolism and to ameliorate metabolic syndrome.

  8. Robert Feulgen Prize Lecture 1999. Detection and amplification systems for sensitive, multiple-target DNA and RNA in situ hybridization: looking inside cells with a spectrum of colors.

    PubMed

    Speel, E J

    1999-08-01

    In situ hybridization (ISH) is a powerful technique for localizing specific nucleic acid sequences (DNA, RNA) in microscopic preparations of tissues, cells, chromosomes, and linear DNA fibers. To date, a wide variety of research and diagnostic applications of ISH have been described, making the technique an integral part of studies concerning gene mapping, gene expression, RNA processing and transport, the three-dimensional organization of the nucleus, tumor genetics, microbial infections, and prenatal diagnosis. In this review, I first describe the ISH procedure in short and then focus on the currently available non-radioactive probe-labeling and cytochemical detection methodologies that are utilized to visualize one or multiple different nucleic acid targets in situ with different colors. Special emphasis is placed on the procedures applying fluorescence and brightfield microscopy, the simultaneous detection of nucleic acids and proteins by combined ISH and immunocytochemistry, and, in addition, on the recent progress that has been made with the introduction of signal amplification procedures to increase the detection sensitivity of ISH. Finally, a comparison of fluorescence, enzyme cytochemical, and colloidal gold silver probe detection systems will be presented, and possible future directions of in situ nucleic acid detection will be discussed. PMID:10460463

  9. Use of a Hierarchical Oligonucleotide Primer Extension Approach for Multiplexed Relative Abundance Analysis of Methanogens in Anaerobic Digestion Systems

    PubMed Central

    Chuang, Hui-Ping; Hsu, Mao-Hsuan; Chen, Wei-Yu

    2013-01-01

    In this study, we established a rapid multiplex method to detect the relative abundances of amplified 16S rRNA genes from known cultivatable methanogens at hierarchical specificities in anaerobic digestion systems treating industrial wastewater and sewage sludge. The method was based on the hierarchical oligonucleotide primer extension (HOPE) technique and combined with a set of 27 primers designed to target the total archaeal populations and methanogens from 22 genera within 4 taxonomic orders. After optimization for their specificities and detection sensitivity under the conditions of multiple single-nucleotide primer extension reactions, the HOPE approach was applied to analyze the methanogens in 19 consortium samples from 7 anaerobic treatment systems (i.e., 513 reactions). Among the samples, the methanogen populations detected with order-level primers accounted for >77.2% of the PCR-amplified 16S rRNA genes detected using an Archaea-specific primer. The archaeal communities typically consisted of 2 to 7 known methanogen genera within the Methanobacteriales, Methanomicrobiales, and Methanosarcinales and displayed population dynamic and spatial distributions in anaerobic reactor operations. Principal component analysis of the HOPE data further showed that the methanogen communities could be clustered into 3 distinctive groups, in accordance with the distribution of the Methanosaeta, Methanolinea, and Methanomethylovorans, respectively. This finding suggested that in addition to acetotrophic and hydrogenotrophic methanogens, the methylotrophic methanogens might play a key role in the anaerobic treatment of industrial wastewater. Overall, the results demonstrated that the HOPE approach is a specific, rapid, and multiplexing platform to determine the relative abundances of targeted methanogens in PCR-amplified 16S rRNA gene products. PMID:24077716

  10. Virus-mediated shRNA knockdown of prodynorphin in the rat nucleus accumbens attenuates depression-like behavior and cocaine locomotor sensitization.

    PubMed

    Cohen, Ami; Whitfield, Timothy W; Kreifeldt, Max; Koebel, Pascale; Kieffer, Brigitte L; Contet, Candice; George, Olivier; Koob, George F

    2014-01-01

    Dynorphins, endogenous opioid peptides that arise from the precursor protein prodynorphin (Pdyn), are hypothesized to be involved in the regulation of mood states and the neuroplasticity associated with addiction. The current study tested the hypothesis that dynorphin in the nucleus accumbens (NAcc) mediates such effects. More specifically, we examined whether knockdown of Pdyn within the NAcc in rats would alter the expression of depressive-like and anxiety-like behavior, as well as cocaine locomotor sensitization. Wistar rats were injected with adeno-associated viral (AAV) vectors encoding either a Pdyn-specific short hairpin RNA (AAV-shPdyn) or a scrambled shRNA (AAV-shScr) as control. Four weeks later, rats were tested for anxiety-like behavior in the elevated plus maze test and depressive-like behavior in the forced swim test (FST). Finally, rats received one daily injection of saline or cocaine (20 mg/kg, i.p.), followed by assessment of locomotion for 4 consecutive days. Following 3 days of abstinence, the rats completed 2 additional daily cocaine/saline locomotor trials. Pdyn knockdown in the NAcc led to a significant reduction in depressive-like behavior in the FST, but had no effect on anxiety-like behavior in the elevated plus maze. Pdyn knockdown did not alter baseline locomotor behavior, the locomotor response to acute cocaine, or the initial sensitization of the locomotor response to cocaine over the first 4 cocaine treatment days. However, following 3 days abstinence the locomotor response to the cocaine challenge returned to their original levels in the AAV-shPdyn rats while remaining heightened in the AAV-shScr rats. These results suggest that dynorphin in a very specific area of the nucleus accumbens contributes to depressive-like states and may be involved in neuroadaptations in the NAcc that contribute to the development of cocaine addiction as a persistent and lasting condition.

  11. Virus-Specific Proteins Synthesized in Cells Infected with RNA+ Temperature-Sensitive Mutants of Sindbis Virus

    PubMed Central

    Scheele, Christina M.; Pfefferkorn, E. R.

    1970-01-01

    All Sindbis virus temperature-sensitive mutants defective in “late” functions were systematically surveyed by acrylamide-gel electrophoresis for similarities and differences in the intracellular pattern of virus-specific proteins synthesized at the permissive and nonpermissive temperatures. Only cells infected with mutants of complementation group C showed an altered pattern. At the nonpermissive temperature, these mutants failed to induce the synthesis of a polypeptide corresponding to the nucleocapsid protein and instead overproduced a protein of higher molecular weight than either viral structural protein. This defect was shown to be irreversible by the finding that 3H-leucine incorporated at 41.5 C specifically failed to appear in the nucleocapsid of virions subsequently released at 29 C. Attempts to demonstrate a precursor protein in wild-type infections were inconclusive. PMID:5461887

  12. Multiplexed Microsphere Suspension Array-Based Immunoassays.

    PubMed

    Lin, Andrew; Salvador, Alexandra; Carter, J Mark

    2015-01-01

    ELISA is an extremely powerful tool to detect analytes because of its sensitivity, selectivity, reproducibility and ease of use. Here we describe sandwich immunoassays performed in suspension on spectrally unique microspheres developed by Luminex. Luminex assays offer the benefit of multiplex analysis of large numbers of analytes in a single reaction. Because the microspheres are spectrally unique, many microspheres, each attached to various antibodies, can be added to a single sample. Luminex instruments can distinguish each microsphere and detect the intensity of a reporter signal for each microsphere. Results are reported in Median Fluorescent Intensities for each analyte. Luminex assays can be used to detect up to 500 analytes in a high-throughput format. Luminex refers to this technology as xMAP(®). Here we describe a routine protocol for a Luminex immunoassay. Other Luminex assays would have to be optimized for specific conditions according to their use. PMID:26160569

  13. Multiplexed Dip Pen Nanolithography patterning by simple desktop nanolithography platform

    NASA Astrophysics Data System (ADS)

    Jang, Jae-Won; Smetana, Alexander; Stiles, Paul

    2010-02-01

    Multiplexed patterning in the micro-scale has been required in order to accomplish functional bio-materials templating on the subcellular length scale. Multiplexed bio-material patterns can be used in several fields: high sensitivity DNA/protein chip development, cell adhesion/differentiation studies, and biological sensor applications. Especially, two or more materials' patterning in subcellular length scale is highly demanding to develop a multi-functional and highintegrated chip device. The multiplexing patterning of two or more materials is a challenge because of difficulty in an alignment and a precision of patterning. In this work, we demonstrate that multiplexed dip pen nanolithography® (DPN®) patterning up to four different material inks by means of using recently developed new generation nanolithography platform (NLP 2000™, NanoInk, Inc., Skokie, IL). Ink materials were prepared by adding different colored fluorescent dyes to matrix carrier materials, such as poly(ethylene glycol) dimethacrylate (PEG-DMA) and lipid material (1,2- dioleoyl-sn-glycero-3-phosphocholine, DOPC). Finally, dot-array patterns of four different inks were obtained in 50 × 50 μm2 area. This lithography platform is capable of patterning 12 separate materials within micrometer areas by efficient use of the available MEMS accessories. This number can be scaled up further with development of new accessories.

  14. MicroRNA-223 Enhances Radiation Sensitivity of U87MG Cells In Vitro and In Vivo by Targeting Ataxia Telangiectasia Mutated

    SciTech Connect

    Liang, Liping; Zhu, Ji; Zaorsky, Nicholas G.; Deng, Yun; Wu, Xingzhong; Liu, Yong; Liu, Fangqi; Cai, Guoxiang; Gu, Weilie; Shen, Lijun; Zhang, Zhen

    2014-03-15

    Purpose: Ataxia telangiectasia mutated (ATM) protein is important in the DNA damage response because it repairs radiation-induced damage in cancers. We examined the effect of microRNA-223 (miR-223), a regulator of ATM expression, on radiation sensitivity of cancer cells. Methods and Materials: Human embryonic kidney 293 T (293T) cells were infected with pLL3.7-miR-223 plasmid to generate the pLL3.7-miR-223 and -empty virus (EV) lentivirus (miR-223 and EV). A dual luciferase assay in which the reporter contained wild-type 3′ untranslated region (UTR) of ATM was performed. U87MG cells were infected with miR-223 or EV to establish the overexpressed stable cell lines (U87-223 or U87-EV, respectively). Cells were irradiated in vitro, and dose enhancement ratios at 2 Gy (DER{sub 2}) were calculated. Hind legs of BALB/c athymic mice were injected with U87-223 or U87-EV cells; after 2 weeks, half of the tumors were irradiated. Tumor volumes were tracked for a total of 5 weeks. Results: The dual luciferase reporter assay showed a significant reduction in luciferase activity of 293T cells cotransfected with miR-223 and the ATM 3′UTR compared to that in EV control. Overexpression of miR-223 in U87MG cells showed that ATM expression was significantly downregulated in the U87-223 cells compared to that in U87-EV (ATM/β-actin mRNA 1.0 vs 1.5, P<.05). U87-223 cells were hypersensitive to radiation compared to U87-EV cells in vitro (DER{sub 2} = 1.32, P<.01). Mice injected with miR-223-expressing tumors had almost the same tumors after 3 weeks (1.5 cm{sup 3} vs 1.7 cm{sup 3}). However, irradiation significantly decreased tumor size in miR-223-expressing tumors compared to those in controls (0.033 cm{sup 3} vs 0.829 cm{sup 3}). Conclusions: miR-223 overexpression downregulates ATM expression and sensitizes U87 cells to radiation in vitro and in vivo. MicroRNA-223 may be a novel cancer-targeting therapy, although its cancer- and patient-specific roles are

  15. Diagnostic evaluation of a multiplexed RT-PCR microsphere array assay for the detection of foot-and-mouth and look-alike disease viruses

    SciTech Connect

    Hindson, B J; Baker, B R; Bentley Tammero, L F; Lenhoff, R J; Naraghi-Arani, P; Vitalis, E A; Slezak, T R; Hullinger, P J; Reid, S M; Ebert, K; Ferris, N P; King, D P

    2007-09-18

    A high-throughput multiplexed assay (Multiplex Version 1.0) was developed for the differential laboratory diagnosis of foot-and-mouth disease virus (FMDV) from viruses which cause clinically similar diseases of livestock. This assay simultaneously screens for five RNA and two DNA viruses using multiplexed reverse transcription PCR (mRT-PCR) amplification coupled with a microsphere hybridization array and flow-cytometric detection. Two of the seventeen primer-probe sets included in this multiplex assay were adopted from previously characterized real-time RT-PCR (rRT-PCR) assays for FMDV. The diagnostic accuracy of the mRT-PCR was evaluated using 287 field samples, including 248 (true positive n= 213, true negative n=34) from suspect cases of foot-and-mouth disease collected from 65 countries between 1965 and 2006 and 39 true negative samples collected from healthy animals. The mRT-PCR assay results were compared with two singleplex rRT-PCR assays, using virus isolation with antigen-ELISA as the reference method. The diagnostic sensitivity of the mRT-PCR assay for FMDV was 93.9% [95% C.I. 89.8-96.4%], compared to 98.1% [95% C.I. 95.3-99.3%] for the two singleplex rRTPCR assays used in combination. In addition, the assay could reliably differentiate between FMDV and other vesicular viruses such as swine vesicular disease virus and vesicular exanthema of swine virus. Interestingly, the mRT-PCR detected parapoxvirus (n=2) and bovine viral diarrhea virus (n=2) in clinical samples, demonstrating the screening potential of this mRT-PCR assay to identify viruses in FMDV-negative material not previously recognized using focused single-target rRT-PCR assays.

  16. Retrospective Species Identification of Microsporidian Spores in Diarrheic Fecal Samples from Human Immunodeficiency Virus/AIDS Patients by Multiplexed Fluorescence In Situ Hybridization▿

    PubMed Central

    Graczyk, Thaddeus K.; Johansson, Michael A.; Tamang, Leena; Visvesvara, Govinda S.; Moura, Laci S.; DaSilva, Alexandre J.; Girouard, Autumn S.; Matos, Olga

    2007-01-01

    In order to assess the applicability of multiplexed fluorescence in situ hybridization (FISH) assay for the clinical setting, we conducted retrospective analysis of 110 formalin-stored diarrheic stool samples from human immunodeficiency virus (HIV)/AIDS patients with intestinal microsporidiosis collected between 1992 and 2003. The multiplexed FISH assay identified microsporidian spores in 94 of 110 (85.5%) samples: 49 (52.1%) were positive for Enterocytozoon bieneusi, 43 (45.8%) were positive for Encephalitozoon intestinalis, 2 (2.1%) were positive for Encephalitozoon hellem, and 9 samples (9.6%) contained both E. bieneusi and E. intestinalis spores. Quantitative spore counts per ml of stool yielded concentration values from 3.5 × 103 to 4.4 × 105 for E. bieneusi (mean, 8.8 × 104/ml), 2.3 × 102 to 7.8 × 104 (mean, 1.5 × 104/ml) for E. intestinalis, and 1.8 × 102 to 3.6 × 102 for E. hellem (mean, 2.7 × 102/ml). Identification of microsporidian spores by multiplex FISH assay was more sensitive than both Chromotrope-2R and CalcoFluor White M2R stains; 85.5% versus 72.7 and 70.9%, respectively. The study demonstrated that microsporidian coinfection in HIV/AIDS patients with intestinal microsporidiosis is not uncommon and that formalin-stored fecal samples older than 10 years may not be suitable for retrospective analysis by techniques targeting rRNA. Multiplexed FISH assay is a reliable, quantitative fluorescence microscopy method for the simultaneous identification of E. bieneusi, E. intestinalis, and E. hellem, as well as Encephalitozoon cuniculi, spores in fecal samples and is a useful tool for assessing spore shedding intensity in intestinal microsporidiosis. The method can be used for epidemiological investigations and applied in clinical settings. PMID:17287331

  17. A Temperature Sensitive Variant of p53 Drives p53-Dependent MicroRNA Expression without Evidence of Widespread Post-Transcriptional Gene Silencing.

    PubMed

    Cabrita, Miguel A; Vanzyl, Erin J; Hamill, Jeff D; Pan, Elysia; Marcellus, Kristen A; Tolls, Victoria J; Alonzi, Rhea C; Pastic, Alyssa; Rambo, Teeghan M E; Sayed, Hadil; McKay, Bruce C

    2016-01-01

    The p53 tumour suppressor is a transcription factor that can regulate the expression of numerous genes including many encoding proteins and microRNAs (miRNAs). The predominant outcomes of a typical p53 response are the initiation of apoptotic cascades and the activation of cell cycle checkpoints. HT29-tsp53 cells express a temperature sensitive variant of p53 and in the absence of exogenous DNA damage, these cells preferentially undergo G1 phase cell cycle arrest at the permissive temperature that correlates with increased expression of the cyclin-dependent kinase inhibitor p21WAF1. Recent evidence also suggests that a variety of miRNAs can induce G1 arrest by inhibiting the expression of proteins like CDK4 and CDK6. Here we used oligonucleotide microarrays to identify p53-regulated miRNAs that are induced in these cells undergoing G1 arrest. At the permissive temperature, the expression of several miRNAs was increased through a combination of either transcriptional or post-transcriptional regulation. In particular, miR-34a-5p, miR-143-3p and miR-145-5p were strongly induced and they reached levels comparable to that of reference miRNAs (miR-191 and miR-103). Importantly, miR-34a-5p and miR-145-5p are known to silence the Cdk4 and/or Cdk6 G1 cyclin-dependent kinases (cdks). Surprisingly, there was no p53-dependent decrease in the expression of either of these G1 cdks. To search for other potential targets of p53-regulated miRNAs, p53-downregulated mRNAs were identified through parallel microarray analysis of mRNA expression. Once again, there was no clear effect of p53 on the repression of mRNAs under these conditions despite a remarkable increase in p53-induced mRNA expression. Therefore, despite a strong p53 transcriptional response, there was no clear evidence that p53-responsive miRNA contributed to gene silencing. Taken together, the changes in cell cycle distribution in this cell line at the permissive temperature is likely attributable to transcriptional

  18. A Temperature Sensitive Variant of p53 Drives p53-Dependent MicroRNA Expression without Evidence of Widespread Post-Transcriptional Gene Silencing

    PubMed Central

    Cabrita, Miguel A.; Vanzyl, Erin J.; Hamill, Jeff D.; Pan, Elysia; Marcellus, Kristen A.; Tolls, Victoria J.; Alonzi, Rhea C.; Pastic, Alyssa; Rambo, Teeghan M. E.; Sayed, Hadil; McKay, Bruce C.

    2016-01-01

    The p53 tumour suppressor is a transcription factor that can regulate the expression of numerous genes including many encoding proteins and microRNAs (miRNAs). The predominant outcomes of a typical p53 response are the initiation of apoptotic cascades and the activation of cell cycle checkpoints. HT29-tsp53 cells express a temperature sensitive variant of p53 and in the absence of exogenous DNA damage, these cells preferentially undergo G1 phase cell cycle arrest at the permissive temperature that correlates with increased expression of the cyclin-dependent kinase inhibitor p21WAF1. Recent evidence also suggests that a variety of miRNAs can induce G1 arrest by inhibiting the expression of proteins like CDK4 and CDK6. Here we used oligonucleotide microarrays to identify p53-regulated miRNAs that are induced in these cells undergoing G1 arrest. At the permissive temperature, the expression of several miRNAs was increased through a combination of either transcriptional or post-transcriptional regulation. In particular, miR-34a-5p, miR-143-3p and miR-145-5p were strongly induced and they reached levels comparable to that of reference miRNAs (miR-191 and miR-103). Importantly, miR-34a-5p and miR-145-5p are known to silence the Cdk4 and/or Cdk6 G1 cyclin-dependent kinases (cdks). Surprisingly, there was no p53-dependent decrease in the expression of either of these G1 cdks. To search for other potential targets of p53-regulated miRNAs, p53-downregulated mRNAs were identified through parallel microarray analysis of mRNA expression. Once again, there was no clear effect of p53 on the repression of mRNAs under these conditions despite a remarkable increase in p53-induced mRNA expression. Therefore, despite a strong p53 transcriptional response, there was no clear evidence that p53-responsive miRNA contributed to gene silencing. Taken together, the changes in cell cycle distribution in this cell line at the permissive temperature is likely attributable to transcriptional

  19. Multiplex RT-PCR and indirect immunofluorescence assays for detection and subtyping of human influenza virus in Tunisia.

    PubMed

    Ben M'hadheb, Manel; Harrabi, Myriam; Souii, Amira; Jrad-Battikh, Nadia; Gharbi, Jawhar

    2015-03-01

    Influenza viruses are negative stranded segmented RNA viruses belonging to Orthomyxoviridae family. They are classified into three types A, B, and C. Type A influenza viruses are classified into subtypes according to the antigenic characters of the surface glycoproteins: hemagglutinin (H) and neuraminidase (N). The aim of the present study is to develop a fast and reliable multiplex RT-PCR technique for detecting simultaneously the subtypes A/H1N1 and A/H3N2 of influenza virus. Our study included 398 patients (mean age 30.33 ± 19.92 years) with flu or flu-like syndromes, consulting physicians affiliated with collaborating teams. A multiplex RT-PCR detecting A/H1N1 and A/H3N2 influenza viruses and an examination by indirect immunofluorescence (IFI) were performed. In the optimized conditions, we diagnosed by IFI a viral infection in 90 patients (22.6 %): 85 cases of influenza type A, four cases of influenza type B, and only one case of coinfection with types A and B. An evaluation of the technique was performed on 19 clinical specimens positive in IFI, and we detected eight cases of A/H3N2, five cases of A/H1N1, one case of influenza virus type A which is not an H1N1 nor H3N2, and five negative cases. Multiplex RT-PCR is a sensitive technique allowing an effective and fast diagnosis of respiratory infections caused by influenza viruses in which the optimization often collides with problems of sensibility.

  20. Genome-wide methylation profiling and a multiplex construction for the identification of body fluids using epigenetic markers.

    PubMed

    Lee, Hwan Young; An, Ja Hyun; Jung, Sang-Eun; Oh, Yu Na; Lee, Eun Young; Choi, Ajin; Yang, Woo Ick; Shin, Kyoung-Jin

    2015-07-01

    The identification of body fluids found at crime scenes can contribute to solving crimes by providing important insights into crime scene reconstruction. In the present study, body fluid-specific epigenetic marker candidates were identified from genome-wide DNA methylation profiling of 42 body fluid samples including blood, saliva, semen, vaginal fluid and menstrual blood using the Illumina Infinium HumanMethylation450 BeadChip array. A total of 64 CpG sites were selected as body fluid-specific marker candidates by having more than 20% discrepancy in DNA methylation status between a certain type of body fluid and other types of body fluids and to have methylation or unmethylation pattern only in a particular type of body fluid. From further locus-specific methylation analysis in additional samples, 1 to 3 CpG sites were selected for each body fluid. Then, a multiplex methylation SNaPshot reaction was constructed to analyze methylation status of 8 body fluid-specific CpG sites. The developed multiplex reaction positively identifies blood, saliva, semen and the body fluid which originates from female reproductive organ in one reaction, and produces successful DNA methylation profiles in aged or mixed samples. Although it remains to be investigated whether this approach is more sensitive, more practical than RNA- or peptide-based assays and whether it can be successfully applied to forensic casework, the results of the present study will be useful for the forensic investigators dealing with body fluid samples. PMID:25796047

  1. Simultaneous Detection of Brown Rot- and Soft Rot-Causing Bacterial Pathogens from Potato Tubers Through Multiplex PCR.

    PubMed

    Ranjan, R K; Singh, Dinesh; Baranwal, V K

    2016-11-01

    Ralstonia solanacearum (Smith) Yabuuchi et al. and Erwinia carotovora subsp. carotovora (Jones) Bergey et al. (Pectobacterium carotovorum subsp. carotovorum) are the two major bacterial pathogens of potato causing brown rot (wilt) and soft rot diseases, respectively, in the field and during storage. Reliable and early detection of these pathogens are keys to avoid occurrence of these diseases in potato crops and reduce yield loss. In the present study, multiplex polymerase chain reaction (PCR) protocol was developed for simultaneous detection of R. solanacearum and E. carotovora subsp. carotovora from potato tubers. A set of oligos targeting the pectatelyase (pel) gene of E. carotovora subsp. carotovora and the universal primers based on 16S r RNA gene of R. solanacearum were used. The standardized multiplex PCR protocol could detect R. solanacearum and E. carotovora subsp. carotovora up to 0.01 and 1.0 ng of genomic DNA, respectively. The protocol was further validated on 96 stored potato tuber samples, collected from different potato-growing states of India, viz. Uttarakhand, Odisha, Meghalaya and Delhi. 53.1 % tuber samples were positive for R. solanacearum, and 15.1 % of samples were positive for E. carotovora subsp. carotovora, and both the pathogens were positive in 26.0 % samples when BIO-PCR was used. This method offers sensitive, specific, reliable and fast detection of two major bacterial pathogens from potato tubers simultaneously, particularly pathogen-free seed certification in large scale. PMID:27480266

  2. Simultaneous Detection of Brown Rot- and Soft Rot-Causing Bacterial Pathogens from Potato Tubers Through Multiplex PCR.

    PubMed

    Ranjan, R K; Singh, Dinesh; Baranwal, V K

    2016-11-01

    Ralstonia solanacearum (Smith) Yabuuchi et al. and Erwinia carotovora subsp. carotovora (Jones) Bergey et al. (Pectobacterium carotovorum subsp. carotovorum) are the two major bacterial pathogens of potato causing brown rot (wilt) and soft rot diseases, respectively, in the field and during storage. Reliable and early detection of these pathogens are keys to avoid occurrence of these diseases in potato crops and reduce yield loss. In the present study, multiplex polymerase chain reaction (PCR) protocol was developed for simultaneous detection of R. solanacearum and E. carotovora subsp. carotovora from potato tubers. A set of oligos targeting the pectatelyase (pel) gene of E. carotovora subsp. carotovora and the universal primers based on 16S r RNA gene of R. solanacearum were used. The standardized multiplex PCR protocol could detect R. solanacearum and E. carotovora subsp. carotovora up to 0.01 and 1.0 ng of genomic DNA, respectively. The protocol was further validated on 96 stored potato tuber samples, collected from different potato-growing states of India, viz. Uttarakhand, Odisha, Meghalaya and Delhi. 53.1 % tuber samples were positive for R. solanacearum, and 15.1 % of samples were positive for E. carotovora subsp. carotovora, and both the pathogens were positive in 26.0 % samples when BIO-PCR was used. This method offers sensitive, specific, reliable and fast detection of two major bacterial pathogens from potato tubers simultaneously, particularly pathogen-free seed certification in large scale.

  3. Genome-wide methylation profiling and a multiplex construction for the identification of body fluids using epigenetic markers.

    PubMed

    Lee, Hwan Young; An, Ja Hyun; Jung, Sang-Eun; Oh, Yu Na; Lee, Eun Young; Choi, Ajin; Yang, Woo Ick; Shin, Kyoung-Jin

    2015-07-01

    The identification of body fluids found at crime scenes can contribute to solving crimes by providing important insights into crime scene reconstruction. In the present study, body fluid-specific epigenetic marker candidates were identified from genome-wide DNA methylation profiling of 42 body fluid samples including blood, saliva, semen, vaginal fluid and menstrual blood using the Illumina Infinium HumanMethylation450 BeadChip array. A total of 64 CpG sites were selected as body fluid-specific marker candidates by having more than 20% discrepancy in DNA methylation status between a certain type of body fluid and other types of body fluids and to have methylation or unmethylation pattern only in a particular type of body fluid. From further locus-specific methylation analysis in additional samples, 1 to 3 CpG sites were selected for each body fluid. Then, a multiplex methylation SNaPshot reaction was constructed to analyze methylation status of 8 body fluid-specific CpG sites. The developed multiplex reaction positively identifies blood, saliva, semen and the body fluid which originates from female reproductive organ in one reaction, and produces successful DNA methylation profiles in aged or mixed samples. Although it remains to be investigated whether this approach is more sensitive, more practical than RNA- or peptide-based assays and whether it can be successfully applied to forensic casework, the results of the present study will be useful for the forensic investigators dealing with body fluid samples.

  4. Modulation of K2P2.1 and K2P10.1 K+ channel sensitivity to carvedilol by alternative mRNA translation initiation

    PubMed Central

    Kisselbach, J; Seyler, C; Schweizer, P A; Gerstberger, R; Becker, R; Katus, H A; Thomas, D

    2014-01-01

    Background and Purpose The β-receptor antagonist carvedilol blocks a range of ion channels. K2P2.1 (TREK1) and K2P10.1 (TREK2) channels are expressed in the heart and regulated by alternative translation initiation (ATI) of their mRNA, producing functionally distinct channel variants. The first objective was to investigate acute effects of carvedilol on human K2P2.1 and K2P10.1 channels. Second, we sought to study ATI-dependent modulation of K2P K+ current sensitivity to carvedilol. Experimental Approach Using standard electrophysiological techniques, we recorded currents from wild-type and mutant K2P2.1 and K2P10.1 channels in Xenopus oocytes and HEK 293 cells. Key Results Carvedilol concentration-dependently inhibited K2P2.1 channels (IC50,oocytes = 20.3 μM; IC50,HEK = 1.6 μM) and this inhibition was frequency-independent. When K2P2.1 isoforms generated by ATI were studied separately in oocytes, the IC50 value for carvedilol inhibition of full-length channels (16.5 μM) was almost 5-fold less than that for the truncated channel variant (IC50 = 79.0 μM). Similarly, the related K2P10.1 channels were blocked by carvedilol (IC50,oocytes = 24.0 μM; IC50,HEK = 7.6 μM) and subject to ATI-dependent modulation of drug sensitivity. Conclusions and Implications Carvedilol targets K2P2.1 and K2P10.1 K+ channels. This previously unrecognized mechanism supports a general role of cardiac K2P channels as antiarrhythmic drug targets. Furthermore, the work reveals that the sensitivity of the cardiac ion channels K2P2.1 and K2P10.1 to block was modulated by alternative mRNA translation initiation. PMID:25168769

  5. Body Fluid Identification Using mRNA Profiling.

    PubMed

    Roeder, Amy D; Haas, Cordula

    2016-01-01

    RNA analysis is a valuable tool for the identification of the forensically relevant body fluids, saliva, blood, menstrual blood, cervicovaginal fluid, and semen. Multiple human mRNA and bacterial RNA markers have been identified for each of these body fluids. RNA and DNA can be coextracted from the same portion of a sample and RNA markers for different body fluids can be multiplexed in a single PCR, thereby maximizing the number of analyses that can be performed with limited sample material.

  6. Hairpin DNA-functionalized gold colloids for the imaging of mRNA in live cells.

    PubMed

    Jayagopal, Ashwath; Halfpenny, Kristin C; Perez, Jonas W; Wright, David W

    2010-07-21

    A strategy is presented for the live cell imaging of messenger RNA using hairpin DNA-functionalized gold nanoparticles (hAuNP). hAuNP improve upon technologies for studying RNA trafficking by their efficient internalization within live cells without transfection reagents, improved resistance to DNase degradation, low cytotoxicity, and the incorporation of hairpin DNA molecular beacons to confer high specificity and sensitivity to the target mRNA sequence. Furthermore, the targeted nanoparticle-beacon construct, once bound to the target mRNA sequence, remains hybridized to the target, enabling spatial and temporal studies of RNA trafficking and downstream analysis. Targeted hAuNP exhibited high specificity for glyceraldehyde 3-phosphate dehydrogenase (GADPH) mRNA in live normal HEp-2 cells and respiratory syncytial virus (RSV) mRNA in live RSV-infected HEp-2 cells with high target to background ratios. Multiplexed fluorescence imaging of distinct mRNAs in live cells and simultaneous imaging of mRNAs with immunofluorescently stained protein targets in fixed cells was enabled by appropriate selection of molecular beacon fluorophores. Pharmacologic analysis suggested that hAuNP were internalized within cells via membrane-nanoparticle interactions. hAuNP are a promising approach for the real-time analysis of mRNA transport and processing in live cells for elucidation of biological processes and disease pathogenesis.

  7. Evaluation of Digital PCR for Absolute RNA Quantification

    PubMed Central

    Sanders, Rebecca; Mason, Deborah J.; Foy, Carole A.; Huggett, Jim F.

    2013-01-01

    Gene expression measurements detailing mRNA quantities are widely employed in molecular biology and are increasingly important in diagnostic fields. Reverse transcription (RT), necessary for generating complementary DNA, can be both inefficient and imprecise, but remains a quintessential RNA analysis tool using qPCR. This study developed a Transcriptomic Calibration Material and assessed the RT reaction using digital (d)PCR for RNA measurement. While many studies characterise dPCR capabilities for DNA quantification, less work has been performed investigating similar parameters using RT-dPCR for RNA analysis. RT-dPCR measurement using three, one-step RT-qPCR kits was evaluated using single and multiplex formats when measuring endogenous and synthetic RNAs. The best performing kit was compared to UV quantification and sensitivity and technical reproducibility investigated. Our results demonstrate assay and kit dependent RT-dPCR measurements differed significantly compared to UV quantification. Different values were reported by different kits for each target, despite evaluation of identical samples using the same instrument. RT-dPCR did not display the strong inter-assay agreement previously described when analysing DNA. This study demonstrates that, as with DNA measurement, RT-dPCR is capable of accurate quantification of low copy RNA targets, but the results are both kit and target dependent supporting the need for calibration controls. PMID:24073259

  8. Parallel multiplex laser feedback interferometry

    SciTech Connect

    Zhang, Song; Tan, Yidong; Zhang, Shulian

    2013-12-15

    We present a parallel multiplex laser feedback interferometer based on spatial multiplexing which avoids the signal crosstalk in the former feedback interferometer. The interferometer outputs two close parallel laser beams, whose frequencies are shifted by two acousto-optic modulators by 2Ω simultaneously. A static reference mirror is inserted into one of the optical paths as the reference optical path. The other beam impinges on the target as the measurement optical path. Phase variations of the two feedback laser beams are simultaneously measured through heterodyne demodulation with two different detectors. Their subtraction accurately reflects the target displacement. Under typical room conditions, experimental results show a resolution of 1.6 nm and accuracy of 7.8 nm within the range of 100 μm.

  9. Pattern formation in multiplex networks

    PubMed Central

    Kouvaris, Nikos E.; Hata, Shigefumi; Guilera, Albert Díaz-

    2015-01-01

    The advances in understanding complex networks have generated increasing interest in dynamical processes occurring on them. Pattern formation in activator-inhibitor systems has been studied in networks, revealing differences from the classical continuous media. Here we study pattern formation in a new framework, namely multiplex networks. These are systems where activator and inhibitor species occupy separate nodes in different layers. Species react across layers but diffuse only within their own layer of distinct network topology. This multiplicity generates heterogeneous patterns with significant differences from those observed in single-layer networks. Remarkably, diffusion-induced instability can occur even if the two species have the same mobility rates; condition which can never destabilize single-layer networks. The instability condition is revealed using perturbation theory and expressed by a combination of degrees in the different layers. Our theory demonstrates that the existence of such topology-driven instabilities is generic in multiplex networks, providing a new mechanism of pattern formation. PMID:26042606

  10. Multiplex characterization of human pathogens including species and antibiotic-resistance gene identification.

    PubMed

    Barisˇ ić, Ivan; Petzka, Josefine; Schoenthaler, Silvia; Vierlinger, Klemens; Noehammer, Christa; Wiesinger-Mayr, Herbert

    2016-01-01

    The efficient medical treatment of infections requires detailed information about the pathogens involved and potential antibiotic-resistance mechanisms. The dramatically increasing incidence of multidrug-resistant bacteria especially highlights the importance of sophisticated diagnostic tests enabling a fast patient-customized therapy. However, the current molecular detection methods are limited to either the detection of species or only a few antibiotic-resistance genes.In this work, we present a human pathogen characterization assay using a rRNA gene microarray identifying 75 species comprising bacteria and fungi. A statistical classifier was developed to facilitate the automated species identification. Additionally, the clinically most important β-lactamases were identified simultaneously in a 100-plex reaction using padlock probes and the same microarray. The specificity and sensitivity of the combined assay was determined using clinical isolates. The detection limit was 10(5) c.f.u. ml(-1), recovering 89 % of the detectable β-lactamase-encoding genes specifically. The total assay time was less than 7 hand the modular character of the antibiotic-resistance detection allows the easy integration of further genetic targets. In summary, we present a fast, highly specific and sensitive multiplex pathogen characterization assay.

  11. Multiplex detection of agricultural pathogens

    DOEpatents

    Siezak, Thomas R.; Gardner, Shea; Torres, Clinton; Vitalis, Elizabeth; Lenhoff, Raymond J.

    2013-01-15

    Described are kits and methods useful for detection of agricultural pathogens in a sample. Genomic sequence information from agricultural pathogens was analyzed to identify signature sequences, e.g., polynucleotide sequences useful for confirming the presence or absence of a pathogen in a sample. Primer and probe sets were designed and optimized for use in a PCR based, multiplexed Luminex assay and/or an array assay to successfully identify the presence or absence of pathogens in a sample.

  12. Multiplex detection of agricultural pathogens

    DOEpatents

    McBride, Mary Teresa; Slezak, Thomas Richard; Messenger, Sharon Lee

    2010-09-14

    Described are kits and methods useful for detection of seven agricultural pathogens (BPSV; BHV; BVD; FMDV; BTV; SVD; and VESV) in a sample. Genomic sequence information from 7 agricultural pathogens was analyzed to identify signature sequences, e.g., polynucleotide sequences useful for confirming the presence or absence of a pathogen in a sample. Primer and probe sets were designed and optimized for use in a PCR based, multiplexed Luminex assay to successfully identify the presence or absence of pathogens in a sample.

  13. Antibody Colocalization Microarray: A Scalable Technology for Multiplex Protein Analysis in Complex Samples*

    PubMed Central

    Pla-Roca, M.; Leulmi, R. F.; Tourekhanova, S.; Bergeron, S.; Laforte, V.; Moreau, E.; Gosline, S. J. C.; Bertos, N.; Hallett, M.; Park, M.; Juncker, D.

    2012-01-01

    DNA microarrays were rapidly scaled up from 256 to 6.5 million targets, and although antibody microarrays were proposed earlier, sensitive multiplex sandwich assays have only been scaled up to a few tens of targets. Cross-reactivity, arising because detection antibodies are mixed, is a known weakness of multiplex sandwich assays that is mitigated by lengthy optimization. Here, we introduce (1) vulnerability as a metric for assays. The vulnerability of multiplex sandwich assays to cross-reactivity increases quadratically with the number of targets, and together with experimental results, substantiates that scaling up of multiplex sandwich assays is unfeasible. We propose (2) a novel concept for multiplexing without mixing named antibody colocalization microarray (ACM). In ACMs, both capture and detection antibodies are physically colocalized by spotting to the same two-dimensional coordinate. Following spotting of the capture antibodies, the chip is removed from the arrayer, incubated with the sample, placed back onto the arrayer and then spotted with the detection antibodies. ACMs with up to 50 targets were produced, along with a binding curve for each protein. The ACM was validated by comparing it to ELISA and to a small-scale, conventional multiplex sandwich assay (MSA). Using ACMs, proteins in the serum of breast cancer patients and healthy controls were quantified, and six candidate biomarkers identified. Our results indicate that ACMs are sensitive, robust, and scalable. PMID:22171321

  14. Detection of adenoviruses and enteroviruses in tap water and river water by reverse transcription multiplex PCR.

    PubMed

    Cho, H B; Lee, S H; Cho, J C; Kim, S J

    2000-05-01

    A reverse transcription (RT) multiplex polymerase chain reaction (PCR) assay was developed to simultaneously detect adenoviruses and enteroviruses, both of which have attracted much attention as molecular indices of viral pollution in environmental samples. The method involves a reverse transcription step, followed by a multiplex nested PCR in which the combination of primers amplifies cDNA from enteroviruses and adenoviruses. The sensitivity of this assay was found to be similar to that of each monoplex PCR or RT-PCR assay, and to be consistent regardless of relative concentrations of adenoviruses and enteroviruses. To assess suitability and environmental application of the RT multiplex PCR assay, a total of 12 river water samples and 4 tap water samples were analyzed by RT multiplex PCR, each monoplex PCR or RT-PCR, and cell culture assay on the Buffalo Green Monkey kidney cell line. The sensitivity of the RT multiplex PCR was also found to be similar to that of each monoplex PCR in environmental samples. This suggests the RT multiplex PCR assay could be applied to the routine monitoring of viral pollution in environmental waters.

  15. Multiplex detection of food allergens and gluten.

    PubMed

    Cho, Chung Y; Nowatzke, William; Oliver, Kerry; Garber, Eric A E

    2015-05-01

    To help safeguard the food supply and detect the presence of undeclared food allergens and gluten, most producers and regulatory agencies rely on commercial test kits. Most of these are ELISAs with a few being PCR-based. These methods are very sensitive and analyte specific, requiring different assays to detect each of the different food allergens. Mass spectrometry offers an alternative approach whereby multiple allergens may be detected simultaneously. However, mass spectrometry requires expensive equipment, highly trained analysts, and several years before a quantitative approach can be achieved. Using multianalyte profiling (xMAP®) technology, a commercial multiplex test kit based on the use of established antibodies was developed for the simultaneous detection of up to 14 different food allergens plus gluten. The assay simultaneously detects crustacean seafood, egg, gluten, milk, peanut, soy, and nine tree nuts (almond, Brazil nut, cashew, coconut, hazelnut, macadamia, pine nut, pistachio, and walnut). By simultaneously performing multiple tests (typically two) for each analyte, this magnetic bead-based assay offers built-in confirmatory analyses without the need for additional resources. Twenty-five of the assays were performed on buffer extracted samples, while five were conducted on samples extracted using reduced-denatured conditions. Thus, complete analysis for all 14 allergens and gluten requires only two wells of a 96-well microtiter plate. This makes it possible to include in a single analytical run up to 48 samples. All 30 bead sets in this multiplex assay detected 5 ng/mL of food allergen and gluten with responses greater than background. In addition, 26 of the bead sets displayed signal/noise ratios of five or greater. The bead-based design makes this 30-plex assay expandable to incorporate new antibodies and capture/detector methodologies by ascribing these new detectors to any of the unassigned bead sets that are commercially available.

  16. Multiplex detection of food allergens and gluten.

    PubMed

    Cho, Chung Y; Nowatzke, William; Oliver, Kerry; Garber, Eric A E

    2015-05-01

    To help safeguard the food supply and detect the presence of undeclared food allergens and gluten, most producers and regulatory agencies rely on commercial test kits. Most of these are ELISAs with a few being PCR-based. These methods are very sensitive and analyte specific, requiring different assays to detect each of the different food allergens. Mass spectrometry offers an alternative approach whereby multiple allergens may be detected simultaneously. However, mass spectrometry requires expensive equipment, highly trained analysts, and several years before a quantitative approach can be achieved. Using multianalyte profiling (xMAP®) technology, a commercial multiplex test kit based on the use of established antibodies was developed for the simultaneous detection of up to 14 different food allergens plus gluten. The assay simultaneously detects crustacean seafood, egg, gluten, milk, peanut, soy, and nine tree nuts (almond, Brazil nut, cashew, coconut, hazelnut, macadamia, pine nut, pistachio, and walnut). By simultaneously performing multiple tests (typically two) for each analyte, this magnetic bead-based assay offers built-in confirmatory analyses without the need for additional resources. Twenty-five of the assays were performed on buffer extracted samples, while five were conducted on samples extracted using reduced-denatured conditions. Thus, complete analysis for all 14 allergens and gluten requires only two wells of a 96-well microtiter plate. This makes it possible to include in a single analytical run up to 48 samples. All 30 bead sets in this multiplex assay detected 5 ng/mL of food allergen and gluten with responses greater than background. In addition, 26 of the bead sets displayed signal/noise ratios of five or greater. The bead-based design makes this 30-plex assay expandable to incorporate new antibodies and capture/detector methodologies by ascribing these new detectors to any of the unassigned bead sets that are commercially available. PMID

  17. Stabilized interrogation and multiplexing techniques for fibre Bragg grating vibration sensors

    NASA Astrophysics Data System (ADS)

    Bang, Hyung-Joon; Jun, Seung-Moon; Kim, Chun-Gon

    2005-03-01

    We demonstrated a simple interrogation system for multiplexed fibre Bragg grating (FBG) sensors in a high-frequency range. A tunable fibre Fabry-Perot (FFP) filter with narrow free spectral range (FSR) was used to simplify the multiplexing demodulator for FBG vibration sensors. A stabilization-controlling unit was also developed for the maintenance of maximum sensitivity of the sensors. In order to verify the performance of the stabilization control unit, we measured the sensitivity of the FBG sensor by changing environmental temperature, and the system showed an average sensitivity of 2.5 nɛRMS Hz-1/2 for a stabilization-controlled case. Finally, multi-point vibration tests using in-line FBG sensors were conducted to validate the multiplexing performance of the FBG system.

  18. Multiplex Electrochemical Immunoassay Using Gold Nanoparticle Probes and Immunochromatographic Strips

    SciTech Connect

    Mao, Xun; Baloda, Meenu; Gurung, Anant; Lin, Yuehe; Liu, Guodong

    2008-10-20

    We describe a multiplex electrochemical immunoassay based on the use of gold nanoparticle (Au-NP) probes and immunochromatographic strips (ISs). The approach takes advantage of the speed and low cost of the conventional IS tests and the high sensitivities of the nanoparticle-based electrochemical immunoassays. Rabbit IgG(R-IgG) and human IgM (H-IgM) were used as model targets for the demonstration of the proof of concept. The Au-NPs based sandwich immunoreactions were performed on the IS, and the captured gold nanoparticle labels on the test zones were determined by highly-sensitive stripping voltammetric measurement of the dissolved gold ions (III) with a carbon paste electrode. The detection limits are 1.0 and 1.5 ng/mL with the linear ranges of 2.5-250 ng/mL for quantitative detection of R-IgG and H-IgM, respectively. The total assay time is around 25 minutes. Such multiplex electrochemical immunoassay could be readily highly multiplexed to allow simultaneous parallel detection of numerous proteins and is expected to open new opportunities for protein diagnostics and biosecurity.

  19. Large-scale fibre-array multiplexing

    SciTech Connect

    Cheremiskin, I V; Chekhlova, T K

    2001-05-31

    The possibility of creating a fibre multiplexer/demultiplexer with large-scale multiplexing without any basic restrictions on the number of channels and the spectral spacing between them is shown. The operating capacity of a fibre multiplexer based on a four-fibre array ensuring a spectral spacing of 0.7 pm ({approx} 10 GHz) between channels is demonstrated. (laser applications and other topics in quantum electronics)

  20. Sensitivity, Specificity, and Clinical Value of Human Papillomavirus (HPV) E6/E7 mRNA Assay as a Triage Test for Cervical Cytology and HPV DNA Test ▿

    PubMed Central

    Benevolo, Maria; Vocaturo, Amina; Caraceni, Donatella; French, Deborah; Rosini, Sandra; Zappacosta, Roberta; Terrenato, Irene; Ciccocioppo, Lucia; Frega, Antonio; Rossi, Paolo Giorgi

    2011-01-01

    There is evidence that testing for human papillomavirus (HPV) E6/E7 mRNA is more specific than testing for HPV DNA. A retrospective study was carried out to evaluate the performance of the PreTect HPV-Proofer E6/E7 mRNA assay (Norchip) as a triage test for cytology and HPV DNA testing. This study analyzed 1,201 women, 688 of whom had a colposcopy follow-up and 195 of whom had histology-confirmed high-grade intraepithelial neoplasia or worse (CIN2+). The proportion of positive results and the sensitivity and specificity for CIN2+ were determined for HPV mRNA in comparison to HPV DNA and cytology. All data were adjusted for follow-up completeness. Stratified by cytological grades, the HPV mRNA sensitivity was 83% (95% confidence interval [CI] = 63 to 94%) in ASC-US (atypical squamous cells of undetermined significance), 62% (95% CI = 47 to 75%) in L-SIL (low-grade squamous intraepithelial lesion), and 67% (95% CI = 57 to 76%) in H-SIL (high-grade squamous intraepithelial lesion). The corresponding figures were 99, 91, and 96%, respectively, for HPV DNA. The specificities were 82, 76, and 45%, respectively, for HPV mRNA and 29, 13, and 4%, respectively, for HPV DNA. Used as a triage test for ASC-US and L-SIL, mRNA reduced colposcopies by 79% (95% CI = 74 to 83%) and 69% (95% CI = 65 to 74%), respectively, while HPV DNA reduced colposcopies by 38% (95% CI = 32 to 44%) and by 15% (95% CI = 12 to 19%), respectively. As a HPV DNA positivity triage test, mRNA reduced colposcopies by 63% (95% CI = 60 to 66%), having 68% sensitivity (95% CI = 61 to 75%), whereas cytology at the ASC-US+ threshold reduced colposcopies by 23% (95% CI = 20 to 26%), showing 92% sensitivity (95% CI = 87 to 95%). In conclusion, PreTect HPV-Proofer mRNA can serve as a better triage test than HPV DNA to reduce colposcopy referral in both ASC-US and L-SIL. It is also more efficient than cytology for the triage of HPV DNA-positive women. Nevertheless, its low sensitivity demands a strict follow-up of

  1. A high-throughput multiplex method adapted for GMO detection.

    PubMed

    Chaouachi, Maher; Chupeau, Gaëlle; Berard, Aurélie; McKhann, Heather; Romaniuk, Marcel; Giancola, Sandra; Laval, Valérie; Bertheau, Yves; Brunel, Dominique

    2008-12-24

    A high-throughput multiplex assay for the detection of genetically modified organisms (GMO) was developed on the basis of the existing SNPlex method designed for SNP genotyping. This SNPlex assay allows the simultaneous detection of up to 48 short DNA sequences (approximately 70 bp; "signature sequences") from taxa endogenous reference genes, from GMO constructions, screening targets, construct-specific, and event-specific targets, and finally from donor organisms. This assay avoids certain shortcomings of multiplex PCR-based methods already in widespread use for GMO detection. The assay demonstrated high specificity and sensitivity. The results suggest that this assay is reliable, flexible, and cost- and time-effective for high-throughput GMO detection.

  2. Measuring and modeling correlations in multiplex networks.

    PubMed

    Nicosia, Vincenzo; Latora, Vito

    2015-09-01

    The interactions among the elementary components of many complex systems can be qualitatively different. Such systems are therefore naturally described in terms of multiplex or multilayer networks, i.e., networks where each layer stands for a different type of interaction between the same set of nodes. There is today a growing interest in understanding when and why a description in terms of a multiplex network is necessary and more informative than a single-layer projection. Here we contribute to this debate by presenting a comprehensive study of correlations in multiplex networks. Correlations in node properties, especially degree-degree correlations, have been thoroughly studied in single-layer networks. Here we extend this idea to investigate and characterize correlations between the different layers of a multiplex network. Such correlations are intrinsically multiplex, and we first study them empirically by constructing and analyzing several multiplex networks from the real world. In particular, we introduce various measures to characterize correlations in the activity of the nodes and in their degree at the different layers and between activities and degrees. We show that real-world networks exhibit indeed nontrivial multiplex correlations. For instance, we find cases where two layers of the same multiplex network are positively correlated in terms of node degrees, while other two layers are negatively correlated. We then focus on constructing synthetic multiplex networks, proposing a series of models to reproduce the correlations observed empirically and/or to assess their relevance. PMID:26465526

  3. Measuring and modeling correlations in multiplex networks

    NASA Astrophysics Data System (ADS)

    Nicosia, Vincenzo; Latora, Vito

    2015-09-01

    The interactions among the elementary components of many complex systems can be qualitatively different. Such systems are therefore naturally described in terms of multiplex or multilayer networks, i.e., networks where each layer stands for a different type of interaction between the same set of nodes. There is today a growing interest in understanding when and why a description in terms of a multiplex network is necessary and more informative than a single-layer projection. Here we contribute to this debate by presenting a comprehensive study of correlations in multiplex networks. Correlations in node properties, especially degree-degree correlations, have been thoroughly studied in single-layer networks. Here we extend this idea to investigate and characterize correlations between the different layers of a multiplex network. Such correlations are intrinsically multiplex, and we first study them empirically by constructing and analyzing several multiplex networks from the real world. In particular, we introduce various measures to characterize correlations in the activity of the nodes and in their degree at the different layers and between activities and degrees. We show that real-world networks exhibit indeed nontrivial multiplex correlations. For instance, we find cases where two layers of the same multiplex network are positively correlated in terms of node degrees, while other two layers are negatively correlated. We then focus on constructing synthetic multiplex networks, proposing a series of models to reproduce the correlations observed empirically and/or to assess their relevance.

  4. Reticulocyte RNA-Dependent RNA Polymerase

    PubMed Central

    Downey, Kathleen M.; Byrnes, John J.; Jurmark, Bonnie S.; So, Antero G.

    1973-01-01

    A cytoplasmic, microsomal bound RNA-dependent RNA polymerase has been purified 2500-fold from rabbit reticulocyte lysates. The synthesis of RNA with the purified enzyme is absolutely dependent on the addition of an RNA template. The best template is hemoglobin messenger RNA, while bacteriophage RNA and poly(A,G) are less active, and DNA is completely inactive as a template. With poly(A,G) as a template, only UTP and CTP are incorporated into polynucleotide chains, indicating that the RNA polymerase is an RNA replicase and not a terminal transferase. With messenger RNA as a template, all four ribonucleoside triphosphates are required for maximal activity. The RNA-dependent RNA polymerase reaction is extremely sensitive to low concentrations of heme, rifamycin AF/013, and ribonuclease and resistant to actinomycin D and DNase. The discovery of RNA-directed RNA synthesis in reticulocytes offers an additional site for control of gene expression in mammalian cells and provides a possible mechanism for amplification of the expression of specific genes. PMID:4519633

  5. Computer assisted multiplex sequencing. Performance report, August 1, 1992--July 15, 1993

    SciTech Connect

    Not Available

    1993-07-01

    The objectives of this project are automation for optimization of multiplex sequencing. We have integrated direct transfer electrophoresis, automated multiplex hybridizations and automated film reading and applied this toward sequencing of E. coli and human DNA. Primers for the directed dideoxy sequence walking and sequence confirmation steps are synthesized to include DNA tags complementary to an alkaline phosphatase conjugate. A higher throughput synthesis device is well along in testing as are new automated hybridization devices. We have developed software for automatically annotating ORFs and databases of precise termini of proteins and RNA.

  6. Multiplex CRISPR/Cas9-based genome engineering from a single lentiviral vector.

    PubMed

    Kabadi, Ami M; Ousterout, David G; Hilton, Isaac B; Gersbach, Charles A

    2014-10-29

    Engineered DNA-binding proteins that manipulate the human genome and transcriptome have enabled rapid advances in biomedical research. In particular, the RNA-guided CRISPR/Cas9 system has recently been engineered to create site-specific double-strand breaks for genome editing or to direct targeted transcriptional regulation. A unique capability of the CRISPR/Cas9 system is multiplex genome engineering by delivering a single Cas9 enzyme and two or more single guide RNAs (sgRNAs) targeted to distinct genomic sites. This approach can be used to simultaneously create multiple DNA breaks or to target multiple transcriptional activators to a single promoter for synergistic enhancement of gene induction. To address the need for uniform and sustained delivery of multiplex CRISPR/Cas9-based genome engineering tools, we developed a single lentiviral system to express a Cas9 variant, a reporter gene and up to four sgRNAs from independent RNA polymerase III promoters that are incorporated into the vector by a convenient Golden Gate cloning method. Each sgRNA is efficiently expressed and can mediate multiplex gene editing and sustained transcriptional activation in immortalized and primary human cells. This delivery system will be significant to enabling the potential of CRISPR/Cas9-based multiplex genome engineering in diverse cell types.

  7. Multiplex CRISPR/Cas9-based genome engineering from a single lentiviral vector.

    PubMed

    Kabadi, Ami M; Ousterout, David G; Hilton, Isaac B; Gersbach, Charles A

    2014-10-29

    Engineered DNA-binding proteins that manipulate the human genome and transcriptome have enabled rapid advances in biomedical research. In particular, the RNA-guided CRISPR/Cas9 system has recently been engineered to create site-specific double-strand breaks for genome editing or to direct targeted transcriptional regulation. A unique capability of the CRISPR/Cas9 system is multiplex genome engineering by delivering a single Cas9 enzyme and two or more single guide RNAs (sgRNAs) targeted to distinct genomic sites. This approach can be used to simultaneously create multiple DNA breaks or to target multiple transcriptional activators to a single promoter for synergistic enhancement of gene induction. To address the need for uniform and sustained delivery of multiplex CRISPR/Cas9-based genome engineering tools, we developed a single lentiviral system to express a Cas9 variant, a reporter gene and up to four sgRNAs from independent RNA polymerase III promoters that are incorporated into the vector by a convenient Golden Gate cloning method. Each sgRNA is efficiently expressed and can mediate multiplex gene editing and sustained transcriptional activation in immortalized and primary human cells. This delivery system will be significant to enabling the potential of CRISPR/Cas9-based multiplex genome engineering in diverse cell types. PMID:25122746

  8. Single-pair fluorescence resonance energy transfer analysis of mRNA transcripts for highly sensitive gene expression profiling in near real time.

    PubMed

    Peng, Zhiyong; Young, Brandon; Baird, Alison E; Soper, Steven A

    2013-08-20

    Expression analysis of mRNAs transcribed from certain genes can be used as important sources of biomarkers for in vitro diagnostics. While the use of reverse transcription quantitative PCR (RT-qPCR) can provide excellent analytical sensitivity for monitoring transcript numbers, more sensitive approaches for expression analysis that can report results in near real-time are needed for many critical applications. We report a novel assay that can provide exquisite limits-of-quantitation and consists of reverse transcription (RT) followed by a ligase detection reaction (LDR) with single-pair fluorescence resonance energy transfer (spFRET) to provide digital readout through molecular counting. For this assay, no PCR was employed, which enabled short assay turnaround times. To facilitate implementation of the assay, a cyclic olefin copolymer (COC) microchip, which was fabricated using hot embossing, was employed to carry out the LDR in a continuous flow format with online single-molecule detection following the LDR. As demonstrators of the assay's utility, MMP-7 mRNA was expression profiled from several colorectal cancer cell lines. It was found that the RT-LDR/spFRET assay produced highly linear calibration plots even in the low copy number regime. Comparison to RT-qPCR indicated a better linearity over the low copy number range investigated (10-10,000 copies) with an R(2) = 0.9995 for RT-LDR/spFRET and R(2) = 0.98 for RT-qPCR. In addition, differentiating between copy numbers of 10 and 50 could be performed with higher confidence using RT-LDR/spFRET. To demonstrate the short assay turnaround times obtainable using the RT-LDR/spFRET assay, a two thermal cycle LDR was carried out on amphiphysin gene transcripts that can serve as important diagnostic markers for ischemic stroke. The ability to supply diagnostic information on possible stroke events in short turnaround times using RT-LDR/spFRET will enable clinicians to treat patients effectively with appropriate time-sensitive

  9. Reducing multiplexing artifacts in multi-pinhole SPECT with a stacked silicon-germanium system: a simulation study.

    PubMed

    Johnson, Lindsay C; Shokouhi, Sepideh; Peterson, Todd E

    2014-12-01

    In pinhole single photon emission computed tomography (SPECT), multi-pinhole collimators can increase sensitivity but may lead to projection overlap, or multiplexing, which can cause image artifacts. In this work, we explore whether a stacked-detector configuration with a germanium and a silicon detector, used with 123I (27-32, 159 keV), where little multiplexing occurs in the Si projections, can reduce image artifacts caused by highly-multiplexed Ge projections. Simulations are first used to determine a reconstruction method that combines the Si and Ge projections to maximize image quality. Next, simulations of different pinhole configurations (varying projection multiplexing) in conjunction with digital phantoms are used to examine whether additional Si projections mitigate artifacts from the multiplexing in the Ge projections. Reconstructed images using both Si and Ge data are compared to those using Ge data alone. Normalized mean-square error and normalized standard deviation provide a quantitative evaluation of reconstructed images' error and noise, respectively, and are used to evaluate the impact of the additional nonmultiplexed data on image quality. For a qualitative comparison, the differential point response function is used to examine multiplexing artifacts. Results show that in cases of highly-multiplexed Ge projections, the addition of low-multiplexed Si projections helps to reduce image artifacts both quantitatively and qualitatively.

  10. Assembling the Streptococcus thermophilus clustered regularly interspaced short palindromic repeats (CRISPR) array for multiplex DNA targeting.

    PubMed

    Guo, Lijun; Xu, Kun; Liu, Zhiyuan; Zhang, Cunfang; Xin, Ying; Zhang, Zhiying

    2015-06-01

    In addition to the advantages of scalable, affordable, and easy to engineer, the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) technology is superior for multiplex targeting, which is laborious and inconvenient when achieved by cloning multiple gRNA expressing cassettes. Here, we report a simple CRISPR array assembling method which will facilitate multiplex targeting usage. First, the Streptococcus thermophilus CRISPR3/Cas locus was cloned. Second, different CRISPR arrays were assembled with different crRNA spacers. Transformation assays using different Escherichia coli strains demonstrated efficient plasmid DNA targeting, and we achieved targeting efficiency up to 95% with an assembled CRISPR array with three crRNA spacers.

  11. Multiplex detection of respiratory pathogens

    DOEpatents

    McBride, Mary; Slezak, Thomas; Birch, James M.

    2012-07-31

    Described are kits and methods useful for detection of respiratory pathogens (influenza A (including subtyping capability for H1, H3, H5 and H7 subtypes) influenza B, parainfluenza (type 2), respiratory syncytial virus, and adenovirus) in a sample. Genomic sequence information from the respiratory pathogens was analyzed to identify signature sequences, e.g., polynucleotide sequences useful for confirming the presence or absence of a pathogen in a sample. Primer and probe sets were designed and optimized for use in a PCR based, multiplexed Luminex assay to successfully identify the presence or absence of pathogens in a sample.

  12. Arthrogryposis multiplex congenita, AD 1156.

    PubMed

    Gordon, E C

    1996-01-01

    A case of arthrogryposis multiplex congenita in an eight-year-old girl was recounted by Thomas of Monmouth in a mid twelfth-century English hagiographic narrative, The Life and Miracles of St William of Norwich. The child had deformities of both hands and both feet at birth, and she developed torticollis and probably had some degree of hypotonia. She needed total care, her family took her to the tomb of St William in the cathedral at Norwich. This visit produced some sort of improvement in her health. Her parents, seeking a miracle, were satisfied that one had occurred. PMID:8606020

  13. Flexible Multiplexed Surface Temperature Sensor

    NASA Technical Reports Server (NTRS)

    Daryabeigi, Kamran; Dillon-Townes, L. A.; Johnson, Preston B.; Ash, Robert L.

    1995-01-01

    Unitary array of sensors measures temperatures at points distributed over designated area on surface. Useful in measuring surface temperatures of aerodynamic models and thermally controlled objects. Made of combination of integrated-circuit microchips and film circuitry. Temperature-sensing chips scanned at speeds approaching 10 kHz. Operating range minus 40 degrees C to 120 degrees C. Flexibility of array conforms to curved surfaces. Multiplexer eliminates numerous monitoring cables. Control of acquisition and recording of data effected by connecting array to microcomputers via suitable interface circuitry.

  14. Detection of RNA viruses: current technologies and future perspectives.

    PubMed

    Cella, Lakshmi N; Blackstock, Daniel; Yates, Marylynn A; Mulchandani, Ashok; Chen, Wilfred

    2013-01-01

    RNA viruses constitute one of the major classes of pathogenic organisms causing human diseases, with varying degrees of severity. This review summarizes the conventional and emerging technologies that are available for the detection of these organisms. Cell culture-based techniques for viral detection have been popular since their inception and continue to be the gold standard against which all other techniques. Over many years, these techniques have undergone some radical changes, reducing the total time needed for detection and improving sensitivity, although even with their reliability and improved features they are being slowly replaced by nucleic acid-based technologies. These molecular detection techniques have revolutionized the area of viral detection by their high sensitivity, selectivity, and short detection time. The majority of nucleic acid-based techniques depend on amplifying viral RNA; however, there are some newer emerging techniques that detect viral RNA in live cells using various configurations of florescent probes. In addition, nucleic acid-based technology has made it possible for multiviral detection with either multiplex polymerase chain reaction assays or microarrays. Every technique described in this review has its own unique abilities, making them indispensable for viral detection. However, we believe that nucleic acid-based technologies will find widespread use after being standardized, limiting other technologies to very specific uses. PMID:23582035

  15. Multiplexed lateral flow biosensors: Technological advances for radically improving point-of-care diagnoses.

    PubMed

    Li, Jia; Macdonald, Joanne

    2016-09-15

    Lateral flow biosensors are a leading technology in point-of-care diagnostics due to their simplicity, rapidness and low cost. Their primacy in this arena continues through technological breakthroughs such as multiplexing: the detection of more than one biomarker in a single assay. Multiplexing capacity is critical for improving diagnostic efficiency, enhancing the diagnostic precision for specific diseases and reducing diagnostic cost. Here we review, for the first time, the various types and strategies employed for creating multiplexed lateral flow biosensors. These are classified into four main categories in terms of specific application or multiplexing level, namely linear, parameter, spatial and conceptual. We describe the practical applications and implications for each approach and compare their advantages and disadvantages. Importantly, multiplexing is still subject to limitations of the traditional lateral flow biosensor, such as sensitivity and specificity. However, by pushing the limitations of the traditional medium into the multiplex arena, several technological breakthroughs are emerging with novel solutions that further expand the utility of lateral flow biosensing for point-of-care applications.

  16. Improved Diagnosis of Periprosthetic Joint Infection by Multiplex PCR of Sonication Fluid from Removed Implants▿

    PubMed Central

    Achermann, Yvonne; Vogt, Markus; Leunig, Michael; Wüst, Jürg; Trampuz, Andrej

    2010-01-01

    The microbiological diagnosis of periprosthetic joint infection (PJI) is crucial for successful antimicrobial treatment. Cultures have limited sensitivity, especially in patients receiving antibiotics. We evaluated the value of multiplex PCR for detection of microbial DNA in sonication fluid from removed orthopedic prostheses. Cases of PJI in which the prosthesis (or part of it) was removed were prospectively included. The removed implant was sonicated, and the resulting sonication fluid was cultured and subjected to multiplex PCR. Of 37 PJI cases (17 hip prostheses, 14 knee prostheses, 4 shoulder prostheses, 1 elbow prosthesis, and 1 ankle prosthesis), pathogens were identified in periprosthetic tissue in 24 (65%) cases, in sonication fluid in 23 (62%) cases, and by multiplex PCR in 29 (78%) cases. The pathogen was detected in 5 cases in sonication fluid only (Propionibacterium acnes in all cases; none of these patients had previously received antibiotics) and in 11 cases by multiplex PCR only (all of these patients had previously received antibiotics). After exclusion of 8 cases caused by P. acnes or Corynebacterium species, which cannot be detected due to the absence of specific primers in the PCR kit, sonication cultures were positive in 17 cases and multiplex PCR sonication cultures were positive in 29 cases (59% versus 100%, respectively; P < 0.01). Among 19 cases (51%) receiving antibiotics, multiplex PCR was positive in all 19 (100%), whereas sonication cultures grew the organism in 8 (42%) (P < 0.01). Multiplex PCR of sonication fluid is a promising test for diagnosis of PJI, particularly in patients who previously received antibiotics. With modified primer sets, multiplex PCR has the potential for further improvement of the diagnosis of PJI. PMID:20164283

  17. SQUID Multiplexers for Cryogenic Detector Arrays

    NASA Technical Reports Server (NTRS)

    Irwin, Kent; Beall, James; Deiker, Steve; Doriese, Randy; Duncan, William; Hilton, Gene; Moseley, S. Harvey; Reintsema, Carl; Stahle, Caroline; Ullom, Joel; Vale, Leila

    2004-01-01

    SQUID multiplexers make it possible to build arrays of thousands of cryogenic detectors with a manageable number of readout channels. We are developing time-division SQUID multiplexers based on Nb trilayer SQUIDs to read arrays of superconducting transition-edge sensors. Our first-generation, 8-channel SQUID multiplexer was used in FIBRE, a one-dimensional TES array for submillimeter astronomy. Our second-generation 32-pixel multiplexer, based on an improved architecture, has been developed for instruments including Constellation-X, SCUBA-2, and solar x-ray astronomy missions. SCUBA-2, which is being developed for the James Clerk Maxwell Telescope, will have more than 10,000 pixels. We are now developing a third-generation architecture based on superconducting hot-electron switches. The use of SQUID multiplexers in instruments operating at above 2 K will also be discussed.

  18. Multiplexing Readout of TES Microcalorimeters Based on Analog Baseband Feedback

    SciTech Connect

    Takei, Y.; Yamasaki, N.Y; Mitsuda, K.; Kimura, S.; Hirakoso, W.; Masui, K.; Korte, P. A. J. de; Kuur, J. van der; Gottardi, L.

    2009-12-16

    A TES microcalorimeter array is a promising spectrometer with excellent energy resolution and a moderate imaging capability. To realize a large format array in space, multiplexing the TES signals at the low tempersture stage is mandatory. We are developing frequency division multiplexing (FDM) based on baseband feedback technique. In FDM, each TES is AC-biased with a different carrier frequency. Signals from several pixels are summed and then read out by one SQUID. The maximum number of multiplexed pixels are limited by the frequency band in which the SQUID can be operated in a flux-locked loop, which is {approx}1 MHz with standard flux-locked loop circuit. In the baseband feedback, the signal ({approx}10 kHz band) from the TES is once demodulated. Then a reconstructed copy of the modulated signal with an appropriate phase is fed back to the SQUID input coil to maintain an approximately constant magnetic flux. This can be implemented even for large cable delays and automatically suppresses the carrier. We developed a prototype electronics for the baseband feedback based on an analog phase sensitive detector (PSD) and a multiplier. Combined with Seiko 80-SSA SQUID amp, open-loop gain of 8 has been obtained for 10 kHz baseband signal at 5 MHz carrier frequency, with a moderate noise contribution of 27pA/{radical}(Hz) at input.

  19. Lauric acid can improve the sensitization of Cetuximab in KRAS/BRAF mutated colorectal cancer cells by retrievable microRNA-378 expression.

    PubMed

    Weng, Wen-Hui; Leung, Wai-Hung; Pang, Yeu-Jye; Hsu, Hsi-Hsien

    2016-01-01

    EGFR-inhibitor (Cetuximab) is one of the main targeted drugs used for metastatic colorectal carcinoma (CRC). The benefit from Cetuximab appears to be limited to a subtype of patients, not for the patients with tumors harboring mutated BRAF or KRAS genes; unfortunately, it accounts for ~40-50% of CRC cases. Previous studies have connected higher expression levels of miR-378 to be commonly presented in patients without BRAF or KRAS mutants than in mutated CRCs. The microRNA-378 (miR-378) is coexpressed with PGC-1β and can be easily induced by fatty acid, for example lauric acid. Therefore, we hypothesized that elevation of miR-378 expression in mutated CRCs may stimulate the cell response to Cetuximab. Herein, seven CRC cell lines with confirmed mutation status were involved in two parallel experiments; directly in vitro transfected miR-378 mimics, and using lauric acid to indirectly induce the level of miR-378 in cells. After the increase of miR-378 in cells by either direct or indirect approaches, sensitivity to Cetuximab was restored in all BRAF mutants (p-value <0.0001-0.0003), and half of KRAS mutants CRC (p-value 0.039-0.007). Further evidence was gained by decreasing expression of MEK and ERK2 proteins after transfection with miR-378; it was similar to the indirect induction by lauric acid approach. In conclusion, the present study demonstrated that lauric acid may efficiently induce miR-378 expression in CRC mutants, and both BRAF and a subtype of KRAS mutants presented significantly improved sensitivity to Cetuximab. Notably, BRAF mutants could even be inhibited in cell proliferation after elevated concentration of miR-378 in cells without combining with targeted therapy. This new approach may shed new light on BRAF or KRAS mutation in CRC patients for clinical trial, since lauric acid may easily be obtain from natural food, and it is supposed to be harmless to the cardiovascular system. PMID:26496897

  20. One-step, multiplexed fluorescence detection of microRNAs based on duplex-specific nuclease signal amplification.

    PubMed

    Yin, Bin-Cheng; Liu, Yu-Qiang; Ye, Bang-Ce

    2012-03-21

    Traditional molecular beacons, widely applied for detection of nucleic acids, have an intrinsic limitation on sensitivity, as one target molecule converts only one beacon molecule to its fluorescent form. Herein, we take advantage of the duplex-specific nuclease (DSN) to create a new signal-amplifying mechanism, duplex-specific nuclease signal amplification (DSNSA), to increase the detection sensitivity of molecular beacons (Taqman probes). DSN nuclease is employed to recycle the process of target-assisted digestion of Taqman probes, thus, resulting in a significant fluorescence signal amplification through which one target molecule cleaves thousands of probe molecules. We further demonstrate the efficiency of this DSNSA strategy for rapid direct quantification of multiple miRNAs in biological samples. Our experimental results showed a quantitative measurement of sequence-specific miRNAs with the detection limit in the femtomolar range, nearly 5 orders of magnitude lower than that of conventional molecular beacons. This amplification strategy also demonstrated a high selectivity for discriminating differences between miRNA family members. Considering the superior sensitivity and specificity, as well as the multiplex and simple-to-implement features, this method promises a great potential of becoming a routine tool for simultaneously quantitative analysis of multiple miRNAs in tissues or cells, and supplies valuable information for biomedical research and clinical early diagnosis.

  1. Messenger RNA profiling for forensic body fluid identification: research and applications.

    PubMed

    Wang, Zheng; Zhang, Su-hua; Di, Zhou; Zhao, Shu-min; Li, Cheng-tao

    2013-10-01

    Identifying the origin of body fluids left at a crime scene can give a significant insight into crime scene reconstruction by supporting a link between sample donors and actual criminal acts. However, the conventional body fluid identification methods are prone to various limitations, such as time consumption, intensive labor, nonparallel manner, varying degrees of sensitivity and limited specificity. Recently, the analysis of cell-specific messenger RNA expression (mRNA profiling) has been proposed to supplant conventional methods for body fluid identification. Since 2011, the collaborative exercises have been organized by the European DNA Profiling Group (EDNAP) in order to evaluate the robustness and reproducibility of mRNA profiling for body fluid identification. The major advantages of mRNA profiling, compared to the conventional methods, include higher sensitivity, greater specificity, the ability of detecting several body fluids in one multiplex reaction, and compatibility with current DNA extraction and analysis procedure. In the current review, we provided an overview of the present knowledge and detection methodologies of mRNA profiling for forensic body fluid identification and discussed its possible practical application to forensic casework. PMID:24466779

  2. Messenger RNA profiling for forensic body fluid identification: research and applications.

    PubMed

    Wang, Zheng; Zhang, Su-hua; Di, Zhou; Zhao, Shu-min; Li, Cheng-tao

    2013-10-01

    Identifying the origin of body fluids left at a crime scene can give a significant insight into crime scene reconstruction by supporting a link between sample donors and actual criminal acts. However, the conventional body fluid identification methods are prone to various limitations, such as time consumption, intensive labor, nonparallel manner, varying degrees of sensitivity and limited specificity. Recently, the analysis of cell-specific messenger RNA expression (mRNA profiling) has been proposed to supplant conventional methods for body fluid identification. Since 2011, the collaborative exercises have been organized by the European DNA Profiling Group (EDNAP) in order to evaluate the robustness and reproducibility of mRNA profiling for body fluid identification. The major advantages of mRNA profiling, compared to the conventional methods, include higher sensitivity, greater specificity, the ability of detecting several body fluids in one multiplex reaction, and compatibility with current DNA extraction and analysis procedure. In the current review, we provided an overview of the present knowledge and detection methodologies of mRNA profiling for forensic body fluid identification and discussed its possible practical application to forensic casework.

  3. Variation-Tolerant Capture and Multiplex Detection of Nucleic Acids: Application to Detection of Microbes

    PubMed Central

    Öhrmalm, Christina; Eriksson, Ronnie; Jobs, Magnus; Simonson, Magnus; Strømme, Maria; Bondeson, Kåre; Herrmann, Björn; Melhus, Åsa

    2012-01-01

    In contrast to ordinary PCRs, which have a limited multiplex capacity and often return false-negative results due to target variation or inhibition, our new detection strategy, VOCMA (variation-tolerant capture multiplex assay), allows variation-tolerant, target-specific capture and detection of many nucleic acids in one test. Here we demonstrate the use of a single-tube, dual-step amplification strategy that overcomes the usual limitations of PCR multiplexing, allowing at least a 22-plex format with retained sensitivity. Variation tolerance was achieved using long primers and probes designed to withstand variation at known sites and a judicious mix of degeneration and universal bases. We tested VOCMA in situations where enrichment from a large sample volume with high sensitivity and multiplexity is important (sepsis; streptococci, enterococci, and staphylococci, several enterobacteria, candida, and the most important antibiotic resistance genes) and where variation tolerance and high multiplexity is important (gastroenteritis; astrovirus, adenovirus, rotavirus, norovirus genogroups I and II, and sapovirus, as well as enteroviruses, which are not associated with gastroenteritis). Detection sensitivities of 10 to 1,000 copies per reaction were achieved for many targets. VOCMA is a highly multiplex, variation-tolerant, general purpose