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1.
Biol Chem ; 404(6): 607-617, 2023 05 25.
Artigo em Inglês | MEDLINE | ID: mdl-36342370

RESUMO

The α2δ3 auxiliary subunit of voltage-activated calcium channels is required for normal synaptic transmission and precise temporal processing of sounds in the auditory brainstem. In mice its loss additionally leads to an inability to distinguish amplitude-modulated tones. Furthermore, loss of function of α2δ3 has been associated with autism spectrum disorder in humans. To investigate possible alterations of network activity in the higher-order auditory system in α2δ3 knockout mice, we analyzed neuronal activity patterns and topography of frequency tuning within networks of the auditory cortex (AC) using two-photon Ca2+ imaging. Compared to wild-type mice we found distinct subfield-specific alterations in the primary auditory cortex, expressed in overall lower correlations between the network activity patterns in response to different sounds as well as lower reliability of these patterns upon repetitions of the same sound. Higher AC subfields did not display these alterations but showed a higher amount of well-tuned neurons along with lower local heterogeneity of the neurons' frequency tuning. Our results provide new insight into AC network activity alterations in an autism spectrum disorder-associated mouse model.


Assuntos
Córtex Auditivo , Transtorno do Espectro Autista , Animais , Humanos , Camundongos , Córtex Auditivo/fisiologia , Transtorno do Espectro Autista/genética , Neurônios , Reprodutibilidade dos Testes , Transmissão Sináptica/fisiologia
2.
Glia ; 64(11): 1892-911, 2016 11.
Artigo em Inglês | MEDLINE | ID: mdl-27458984

RESUMO

Astrocytes form large gap junctional networks that contribute to ion and neurotransmitter homeostasis. Astrocytes concentrate in the lateral superior olive (LSO), a prominent auditory brainstem center. Compared to the LSO, astrocyte density is lower in the region dorsal to the LSO (dLSO) and in the internuclear space between the LSO, the superior paraolivary nucleus (SPN). We questioned whether astrocyte networks exhibit certain properties that reflect the precise neuronal arrangement. Employing whole-cell patch-clamp and concomitant injection of a gap junction-permeable tracer, we analyzed size and orientation of astrocyte networks in LSO, dLSO, and SPN-LSO in acute brainstem slices of mice at postnatal days 10-20. The majority of LSO networks exhibited an oval topography oriented orthogonally to the tonotopic axis, whereas dLSO networks showed no preferred orientation. This correlated with the overall astrocyte morphology in both regions, i.e. LSO astrocyte processes were oriented mainly orthogonally to the tonotopic axis. To assess the spread of small ions within LSO networks, we analyzed the diffusion of Na(+) signals between cells using Na(+) imaging. We found that Na(+) not only diffused between SR101(+) astrocytes, but also from astrocytes into SR101(-) cells. Using PLP-GFP mice for tracing, we could show that LSO networks contained astrocytes and oligodendrocytes. Together, our results demonstrate that LSO astrocytes and LSO oligodendrocytes form functional anisotropic panglial networks that are oriented predominantly orthogonally to the tonotopic axis. Thus, our results point toward an anisotropic ion and metabolite diffusion and a limited glial crosstalk between neighboring isofrequency bands in the LSO. GLIA 2016;64:1892-1911.


Assuntos
Astrócitos/fisiologia , Rede Nervosa/fisiologia , Complexo Olivar Superior/citologia , Potenciais de Ação/efeitos dos fármacos , Animais , Animais Recém-Nascidos , Astrócitos/citologia , Astrócitos/metabolismo , Biotina/análogos & derivados , Biotina/metabolismo , Conexina 30/metabolismo , Conexina 43/metabolismo , Feminino , Junções Comunicantes/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Proteínas da Membrana Plasmática de Transporte de Glicina/metabolismo , Hipocampo/citologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteína Proteolipídica de Mielina/genética , Proteína Proteolipídica de Mielina/metabolismo , Rede Nervosa/citologia , Oligodendroglia/fisiologia , Sódio/metabolismo
3.
eNeuro ; 11(5)2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38627066

RESUMO

Autism spectrum disorder (ASD) is often associated with social communication impairments and specific sound processing deficits, for example, problems in following speech in noisy environments. To investigate underlying neuronal processing defects located in the auditory cortex (AC), we performed two-photon Ca2+ imaging in FMR1 (fragile X messenger ribonucleoprotein 1) knock-out (KO) mice, a model for fragile X syndrome (FXS), the most common cause of hereditary ASD in humans. For primary AC (A1) and the anterior auditory field (AAF), topographic frequency representation was less ordered compared with control animals. We additionally analyzed ensemble AC activity in response to various sounds and found subfield-specific differences. In A1, ensemble correlations were lower in general, while in secondary AC (A2), correlations were higher in response to complex sounds, but not to pure tones. Furthermore, sound specificity of ensemble activity was decreased in AAF. Repeating these experiments 1 week later revealed no major differences regarding representational drift. Nevertheless, we found subfield- and genotype-specific changes in ensemble correlation values between the two times points, hinting at alterations in network stability in FMR1 KO mice. These detailed insights into AC network activity and topography in FMR1 KO mice add to the understanding of auditory processing defects in FXS.


Assuntos
Córtex Auditivo , Modelos Animais de Doenças , Proteína do X Frágil da Deficiência Intelectual , Síndrome do Cromossomo X Frágil , Camundongos Knockout , Animais , Córtex Auditivo/fisiopatologia , Síndrome do Cromossomo X Frágil/fisiopatologia , Síndrome do Cromossomo X Frágil/genética , Proteína do X Frágil da Deficiência Intelectual/genética , Masculino , Camundongos Endogâmicos C57BL , Estimulação Acústica , Percepção Auditiva/fisiologia , Camundongos , Cálcio/metabolismo
4.
Front Neural Circuits ; 17: 1210057, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37521334

RESUMO

The auditory cortex (AC) modulates the activity of upstream pathways in the auditory brainstem via descending (corticofugal) projections. This feedback system plays an important role in the plasticity of the auditory system by shaping response properties of neurons in many subcortical nuclei. The majority of layer (L) 5 corticofugal neurons project to the inferior colliculus (IC). This corticocollicular (CC) pathway is involved in processing of complex sounds, auditory-related learning, and defense behavior. Partly due to their location in deep cortical layers, CC neuron population activity patterns within neuronal AC ensembles remain poorly understood. We employed two-photon imaging to record the activity of hundreds of L5 neurons in anesthetized as well as awake animals. CC neurons are broader tuned than other L5 pyramidal neurons and display weaker topographic order in core AC subfields. Network activity analyses revealed stronger clusters of CC neurons compared to non-CC neurons, which respond more reliable and integrate information over larger distances. However, results obtained from secondary auditory cortex (A2) differed considerably. Here CC neurons displayed similar or higher topography, depending on the subset of neurons analyzed. Furthermore, specifically in A2, CC activity clusters formed in response to complex sounds were spatially more restricted compared to other L5 neurons. Our findings indicate distinct network mechanism of CC neurons in analyzing sound properties with pronounced subfield differences, demonstrating that the topography of sound-evoked responses within AC is neuron-type dependent.


Assuntos
Córtex Auditivo , Colículos Inferiores , Animais , Córtex Auditivo/fisiologia , Vias Auditivas/fisiologia , Colículos Inferiores/fisiologia , Neurônios/fisiologia , Células Piramidais , Estimulação Acústica
5.
Front Neural Circuits ; 17: 1307283, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-38107610

RESUMO

Auditory brainstem neurons in the lateral superior olive (LSO) receive excitatory input from the ipsilateral cochlear nucleus (CN) and inhibitory transmission from the contralateral CN via the medial nucleus of the trapezoid body (MNTB). This circuit enables sound localization using interaural level differences. Early studies have observed an additional inhibitory input originating from the ipsilateral side. However, many of its details, such as its origin, remained elusive. Employing electrical and optical stimulation of afferents in acute mouse brainstem slices and anatomical tracing, we here describe a glycinergic projection to LSO principal neurons that originates from the ipsilateral CN. This inhibitory synaptic input likely mediates inhibitory sidebands of LSO neurons in response to acoustic stimulation.


Assuntos
Núcleo Coclear , Localização de Som , Complexo Olivar Superior , Animais , Camundongos , Complexo Olivar Superior/fisiologia , Núcleo Coclear/fisiologia , Núcleo Olivar/fisiologia , Localização de Som/fisiologia , Neurônios/fisiologia , Vias Auditivas/fisiologia
6.
Clin Chem ; 58(11): 1546-56, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-22923452

RESUMO

BACKGROUND: The majority of established techniques for monitoring real-time PCR amplification involve individual target-specific fluorogenic probes. For analysis of numerous different targets the synthesis of these probes contributes to the overall cost during assay development. Sequence-dependent universal detection techniques overcome this drawback but are prone to detection of unspecific amplification products. We developed the mediator probe PCR as a solution to these problems. METHODS: A set of label-free sequence-specific primary probes (mediator probes), each comprising a target-specific region and a standardized mediator tag, is cleaved upon annealing to its target sequence by the polymerases' 5' nuclease activity. Release of a mediator triggers signal generation by cleavage of a complementary fluorogenic reporter probe. RESULTS: Real-time PCR amplification of human papillomavirus 18 (HPV18), Staphylococcus aureus, Escherichia coli, and Homo sapiens DNA dilution series showed exceptional linearity when detected either by novel mediator probes (r(2) = 0.991-0.999) or state-of-the-art hydrolysis probes (TaqMan probes) (r(2) = 0.975-0.993). For amplification of HPV18 DNA the limits of detection were 78.3 and 85.1 copies per 10-µL reaction when analyzed with the mediator probe and hydrolysis probe, respectively. Duplex amplification of HPV18 target DNA and internal standard had no effects on back calculation of target copy numbers when quantified with either the mediator probe PCR (r(2) = 0.998) or the hydrolysis probe PCR (r(2) = 0.988). CONCLUSIONS: The mediator probe PCR has equal performance to hydrolysis probe PCR and has reduced costs because of the use of universal fluorogenic reporters.


Assuntos
Primers do DNA , DNA/genética , Corantes Fluorescentes , Reação em Cadeia da Polimerase em Tempo Real/métodos , DNA Bacteriano/genética , DNA Viral/genética , Escherichia coli/genética , Fluorescência , Papillomavirus Humano 18/genética , Humanos , Limite de Detecção , Reação em Cadeia da Polimerase em Tempo Real/economia , Staphylococcus aureus/genética , Taq Polimerase
7.
Micromachines (Basel) ; 12(2)2021 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-33562822

RESUMO

This paper presents a universal point-of-care system for fully automated quantification of human T-cell lymphotropic virus type 1 (HTLV-1) proviral load, including genomic RNA, based on digital reverse RNA transcription and c-DNA amplification by MD LAMP (mediator displacement loop-mediated isothermal amplification). A disposable microfluidic LabDisk with pre-stored reagents performs automated nucleic acid extraction, reaction setup, emulsification, reverse transcription, digital DNA amplification, and quantitative fluorogenic endpoint detection with universal reporter molecules. Automated nucleic acid extraction from a suspension of HTLV-1-infected CD4+ T-lymphocytes (MT-2 cells) yielded 8 ± 7 viral nucleic acid copies per MT-2 cell, very similar to the manual reference extraction (7 ± 2 nucleic acid copies). Fully automated sample processing from whole blood spiked with MT-2 cells showed a comparable result of 7 ± 3 copies per MT-2 cell after a run time of two hours and 10 min.

8.
Lab Chip ; 10(19): 2519-26, 2010 Oct 07.
Artigo em Inglês | MEDLINE | ID: mdl-20607174

RESUMO

We present a novel process flow enabling prototyping of microfluidic cartridges made out of polymer films. Its high performance is proven by implementation of a microfluidic genotyping assay testing 22 DNA samples including clinical isolates from patients infected by methicilin-resistant Staphylococcus aureus (MRSA). The microfluidic cartridges (disks) are fabricated by a novel process called microthermoforming by soft lithography (microTSL). Positive moulds are applied allowing for higher moulding precision and very easy demoulding when compared to conventional microthermoforming. High replication accuracies with geometric disk-to-disk variations of less than 1% are typical. We describe and characterise fabrication and application of microfluidic cartridges with wall thicknesses <188 microm thus enabling efficient thermocycling during real-time polymerase chain reaction (PCR). The microfluidic cartridges are designed for operation in a slightly modified commercial thermocycling instrument. This approach demonstrates new opportunities for both microfluidic developments and well-established laboratory instruments. The microfluidic protocol is controlled by centrifugal forces and divides the liquid sample parallely into independent aliquots of 9.8 microl (CV 3.4%, N = 32 wells). The genotyping assays are performed with pre-stored primers and probes for real-time PCR showing a limit of detection well below 10 copies of DNA per reaction well (N = 24 wells in 3 independent disks). The system was evaluated by 44 genotyping assays comprising 22 DNA samples plus duplicates in a total of 11 disks. The samples contained clinical samples of seven different genotypes of MRSA as well as positive and negative controls. The results are in excellent agreement with the reference in microtubes.


Assuntos
Centrifugação/instrumentação , Membranas Artificiais , Staphylococcus aureus Resistente à Meticilina/genética , Técnicas Analíticas Microfluídicas/instrumentação , Polímeros/síntese química , Reação em Cadeia da Polimerase Via Transcriptase Reversa/instrumentação , Desenho de Equipamento , Análise de Falha de Equipamento , Genótipo , Staphylococcus aureus Resistente à Meticilina/isolamento & purificação , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
9.
Lab Chip ; 20(14): 2549-2561, 2020 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-32568322

RESUMO

We present an automated point-of-care testing (POCT) system for rapid detection of species- and resistance markers in methicillin-resistant Staphylococcus aureus (MRSA) at the level of single cells, directly from nasal swab samples. Our novel system allows clear differentiation between MRSA, methicillin-sensitive S. aureus (MSSA) and methicillin-resistant coagulase-negative staphylococci (MR-CoNS), which is not the case for currently used real-time quantitative PCR based systems. On top, the novel approach outcompetes the culture-based methods in terms of its short time-to-result (1 h vs. up to 60 h) and reduces manual labor. The walk-away test is fully automated on the centrifugal microfluidic LabDisk platform. The LabDisk cartridge comprises the unit operations swab-uptake, reagent pre-storage, distribution of the sample into 20 000 droplets, specific enzymatic lysis of Staphylococcus spp. and recombinase polymerase amplification (RPA) of species (vicK) - and resistance (mecA) -markers. LabDisk actuation, incubation and multi-channel fluorescence detection is demonstrated with a clinical isolate and spiked nasal swab samples down to a limit of detection (LOD) of 3 ± 0.3 CFU µl-1 for MRSA. The novel approach of the digital single cell detection is suggested to improve hospital admission screening, timely decision making, and goal-oriented antibiotic therapy. The implementation of a higher degree of multiplexing is required to translate the results into clinical practice.


Assuntos
Staphylococcus aureus Resistente à Meticilina , Infecções Estafilocócicas , Proteínas de Bactérias , Humanos , Staphylococcus aureus Resistente à Meticilina/genética , Testes Imediatos , Infecções Estafilocócicas/diagnóstico , Staphylococcus aureus/genética
10.
Mol Brain ; 11(1): 4, 2018 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-29370841

RESUMO

Neuronal inhibition is mediated by glycine and/or GABA. Inferior colliculus (IC) neurons receive glycinergic and GABAergic inputs, whereas inhibition in hippocampus (HC) predominantly relies on GABA. Astrocytes heterogeneously express neurotransmitter transporters and are expected to adapt to the local requirements regarding neurotransmitter homeostasis. Here we analyzed the expression of inhibitory neurotransmitter transporters in IC and HC astrocytes using whole-cell patch-clamp and single-cell reverse transcription-PCR. We show that most astrocytes in both regions expressed functional glycine transporters (GlyTs). Activation of these transporters resulted in an inward current (IGly) that was sensitive to the competitive GlyT1 agonist sarcosine. Astrocytes exhibited transcripts for GlyT1 but not for GlyT2. Glycine did not alter the membrane resistance (RM) arguing for the absence of functional glycine receptors (GlyRs). Thus, IGly was mainly mediated by GlyT1. Similarly, we found expression of functional GABA transporters (GATs) in all IC astrocytes and about half of the HC astrocytes. These transporters mediated an inward current (IGABA) that was sensitive to the competitive GAT-1 and GAT-3 antagonists NO711 and SNAP5114, respectively. Accordingly, transcripts for GAT-1 and GAT-3 were found but not for GAT-2 and BGT-1. Only in hippocampal astrocytes, GABA transiently reduced RM demonstrating the presence of GABAA receptors (GABAARs). However, IGABA was mainly not contaminated by GABAAR-mediated currents as RM changes vanished shortly after GABA application. In both regions, IGABA was stronger than IGly. Furthermore, in HC the IGABA/IGly ratio was larger compared to IC. Taken together, our results demonstrate that astrocytes are heterogeneous across and within distinct brain areas. Furthermore, we could show that the capacity for glycine and GABA uptake varies between both brain regions.


Assuntos
Astrócitos/metabolismo , Proteínas da Membrana Plasmática de Transporte de GABA/metabolismo , Proteínas da Membrana Plasmática de Transporte de Glicina/metabolismo , Hipocampo/metabolismo , Animais , Glicina/farmacologia , Colículos Inferiores , Ativação do Canal Iônico/efeitos dos fármacos , Cinética , Camundongos Endogâmicos C57BL , Análise de Célula Única , Ácido gama-Aminobutírico/farmacologia
11.
Front Cell Neurosci ; 12: 431, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30542265

RESUMO

Astrocytes and oligodendrocytes in different brain regions form panglial networks and the topography of such networks can correlate with neuronal topography and function. Astrocyte-oligodendrocyte networks in the lateral superior olive (LSO)-an auditory brainstem nucleus-were found to be anisotropic with a preferred orientation orthogonally to the tonotopic axis. We hypothesized that such a specialization might be present in other tonotopically organized brainstem nuclei, too. Thus, we analyzed gap junctional coupling in the center of the inferior colliculus (IC)-another nucleus of the auditory brainstem that exhibits tonotopic organization. In acute brainstem slices obtained from mice, IC networks were traced employing whole-cell patch-clamp recordings of single sulforhodamine (SR) 101-identified astrocytes and concomitant intracellular loading of the gap junction-permeable tracer neurobiotin. The majority of dye-coupled networks exhibited an oval topography, which was preferentially oriented orthogonal to the tonotopic axis. Astrocyte processes showed preferentially the same orientation indicating a correlation between astrocyte and network topography. In addition to SR101-positive astrocytes, IC networks contained oligodendrocytes. Using Na+ imaging, we analyzed the capability of IC networks to redistribute small ions. Na+ bi-directionally diffused between SR101-positive astrocytes and SR101-negative cells-presumably oligodendrocytes-showing the functionality of IC networks. Taken together, our results demonstrate that IC astrocytes and IC oligodendrocytes form functional anisotropic panglial networks that are preferentially oriented orthogonal to the tonotopic axis. Thus, our data indicate that the topographic specialization of glial networks seen in IC and LSO might be a general feature of tonotopically organized auditory brainstem nuclei.

12.
Biotechniques ; 61(3): 123-8, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27625206

RESUMO

Mediator probe (MP) PCR is a real-time PCR approach that uses standardized universal fluorogenic reporter oligonucleotides (UR) in conjunction with label-free sequence-specific probes. To enable multiplex real-time MP PCR, we designed a set of five optimized URs with different fluorescent labels. Performance of the optimized URs was verified in multiplex real-time MP PCR for the detection of a pentaplex food panel and a quadruplex methicillin-resistant Staphylococcus aureus (MRSA) panel. Results were comparable to corresponding multiplex hydrolysis probe (HP) PCR, also designated as TaqMan PCR. Analyses of MRSA DNA standards and DNA extracted from patient swab samples showed improved lower limits of detection (LoDs) by a factor of 2-5 when using quadruplex real-time MP PCR instead of HP PCR. The novel set of standardized URs we present here simplifies development of multiplex real-time PCR assays by requiring only the design of label-free probes. In the future, real-time PCR master mixes could be augmented with up to five standardized fluorogenic URs, each emitting light at a different wavelength.


Assuntos
Corantes Fluorescentes/química , Análise de Alimentos/métodos , Tipagem Molecular/métodos , Reação em Cadeia da Polimerase Multiplex/métodos , Animais , DNA/análise , DNA/classificação , DNA/genética , DNA/isolamento & purificação , Alimentos/classificação , Limite de Detecção , Staphylococcus aureus Resistente à Meticilina/genética , Staphylococcus aureus Resistente à Meticilina/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real/métodos , Reprodutibilidade dos Testes
13.
Lab Chip ; 16(1): 208-16, 2016 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-26610263

RESUMO

Existing systems for digital droplet PCR (ddPCR) either suffer from low integration or are difficult to introduce to mass fabrication. Here we present an integrated system that is compatible to mass fabrication and combines emulsification, PCR, and fluorescence readout in a single chamber within a disposable cartridge (disk). Droplets are generated by injecting the sample into fluorinated oil via centrifugal step emulsification. The resulting emulsion is aligned in the PCR and readout zone by capillary action. During thermocycling, gas bubbles generated by degassing are removed by capillary driven transport through tapered regions in the PCR chamber. Thereby, the positioning of the emulsion within the readout zone of the PCR chamber is ensured at any time and no bubbles are present during readout. Manual handling of the disk solely requires pipetting of oil and PCR mix into the inlet structures, placing the disk into the thermocycler and subsequently into a microarray scanner. The functionality of the ddPCR process chain is demonstrated by quantitative detection of the cystic fibrosis causing mutation p.Phe508del, which is of interest for non-invasive prenatal testing (NIPT). The mutation was detected in a concentration range spanning four orders of magnitude. We envision that this work will lay the base for the development of highly integrated sample-to-digital-answer PCR systems that can be employed in routine clinical diagnosis.


Assuntos
Análise Mutacional de DNA/instrumentação , Reação em Cadeia da Polimerase/instrumentação , Fibrose Cística/diagnóstico , Fibrose Cística/genética , DNA/genética , Emulsões/química , Técnicas Analíticas Microfluídicas/instrumentação , Tamanho da Partícula , Propriedades de Superfície
14.
Lab Chip ; 15(13): 2759-66, 2015 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-25947077

RESUMO

Aqueous microdroplets provide miniaturized reaction compartments for numerous chemical, biochemical or pharmaceutical applications. We introduce centrifugal step emulsification for the fast and easy production of monodisperse droplets. Homogenous droplets with pre-selectable diameters in a range from 120 µm to 170 µm were generated with coefficients of variation of 2-4% and zero run-in time or dead volume. The droplet diameter depends on the nozzle geometry (depth, width, and step size) and interfacial tensions only. Droplet size is demonstrated to be independent of the dispersed phase flow rate between 0.01 and 1 µl s(-1), proving the robustness of the centrifugal approach. Centrifugal step emulsification can easily be combined with existing centrifugal microfluidic unit operations, is compatible to scalable manufacturing technologies such as thermoforming or injection moulding and enables fast emulsification (>500 droplets per second and nozzle) with minimal handling effort (2-3 pipetting steps). The centrifugal microfluidic droplet generation was used to perform the first digital droplet recombinase polymerase amplification (ddRPA). It was used for absolute quantification of Listeria monocytogenes DNA concentration standards with a total analysis time below 30 min. Compared to digital droplet polymerase chain reaction (ddPCR), with processing times of about 2 hours, the overall processing time of digital analysis was reduced by more than a factor of 4.


Assuntos
Emulsões/química , Técnicas de Amplificação de Ácido Nucleico , Ácidos Nucleicos/análise , DNA Bacteriano/análise , Listeria monocytogenes/genética , Técnicas Analíticas Microfluídicas/instrumentação , Recombinases/metabolismo
15.
Biotechniques ; 57(3): 151-5, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25209050

RESUMO

A method for conserving primers and differently labeled fluorogenic hydrolysis (i.e., TaqMan) probes at ambient conditions is presented. Primers and hydrolysis probes with four different fluorophore-quencher combinations (6- FAM-BHQ1, HEX-BHQ1, ROX-BHQ650, and Cy5-BHQ2) were mixed with trehalose and xanthan at final concentrations of 56 mM and 2.78 mM, respectively. Mixtures were air-dried at 23°C for 30 min on strips composed of cyclo olefin polymer (COP), a material widely used in the manufacturing of in vitro diagnostic (IVD) test carriers. After one year of storage, the functionality of the primers and fluorophore-quencher combinations was validated by real-time polymerase chain reaction (real-time PCR), confirming their stability when stored in the presence of stabilizers, with the best results achieved using trehalose. This approach could be of great benefit for manufacturing IVD systems, for example, for genotyping applications based on multiplexing using different fluorescent dyes.


Assuntos
Primers do DNA/química , Excipientes/química , Polissacarídeos Bacterianos/química , Reação em Cadeia da Polimerase em Tempo Real/métodos , Trealose/química , Corantes Fluorescentes , Hidrólise , Reprodutibilidade dos Testes
16.
Methods Mol Biol ; 1160: 55-73, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24740221

RESUMO

Mediator probe PCR (MP PCR) is a novel detection format for real-time nucleic acid analysis. Label-free mediator probes (MP) and fluorogenic universal reporter (UR) oligonucleotides are combined to accomplish signal generation. Compared to conventional hydrolysis probe PCRs costs can thus be saved by using the same fluorogenic UR for signal generation in different assays. This tutorial provides a practical guideline to MP and UR design. MP design rules are very similar to those of hydrolysis probes. The major difference is in the replacement of the fluorophore and quencher by one UR-specific sequence tag, the mediator. Further protocols for the setup of reactions, to detect either DNA or RNA targets with clinical diagnostic target detection as models, are explained. Ready to use designs for URs are suggested and guidelines for their de novo design are provided as well, including a protocol for UR signal generation characterization.


Assuntos
Corantes Fluorescentes/química , Reação em Cadeia da Polimerase em Tempo Real/métodos , Sequência de Bases , DNA Viral/química , DNA Viral/genética , Conformação de Ácido Nucleico , Hibridização de Ácido Nucleico
17.
Lab Chip ; 12(17): 3177-83, 2012 Sep 07.
Artigo em Inglês | MEDLINE | ID: mdl-22767208

RESUMO

Optical gradient forces generated by fast steerable optical tweezers are highly effective for sorting small populations of cells in a lab-on-a-chip environment. The presented system can sort a broad range of different biological specimens by an automated optimisation of the tweezer path and velocity profile. The optimal grab positions for subsequent trap and cell displacements are estimated from the intensity of the bright field image, which is derived theoretically and proven experimentally. We exhibit rapid displacements of 2 µm small mitochondria, yeast cells, rod-shaped bacteria and 30 µm large protoplasts. Reliable sorting of yeast cells in a microfluidic chamber by both morphological criteria and by fluorescence emission is demonstrated.


Assuntos
Separação Celular/métodos , Técnicas Analíticas Microfluídicas/instrumentação , Pinças Ópticas , Animais , Bactérias/isolamento & purificação , Separação Celular/instrumentação , Camundongos , Técnicas Analíticas Microfluídicas/métodos , Mitocôndrias/fisiologia , Protoplastos/citologia , Saccharomyces cerevisiae/isolamento & purificação
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