Dataset Information

Nitrogen Cycle Evaluation (NiCE) Chip for Simultaneous Analysis of Multiple N Cycle-Associated Genes.

ABSTRACT: Various microorganisms play key roles in the nitrogen (N) cycle. Quantitative PCR (qPCR) and PCR amplicon sequencing of N cycle functional genes allow us to analyze the abundance and diversity of microbes responsible for N-transforming reactions in various environmental samples. However, analysis of multiple target genes can be cumbersome and expensive. PCR-independent analysis, such as metagenomics and metatranscriptomics, is useful but expensive, especially when we analyze multiple samples and try to detect N cycle functional genes present at a relatively low abundance. Here, we present the application of microfluidic qPCR chip technology to simultaneously quantify and prepare amplicon sequence libraries for multiple N cycle functional genes as well as taxon-specific 16S rRNA gene markers for many samples. This approach, named the nitrogen cycle evaluation (NiCE) chip, was evaluated by using DNA from pure and artificially mixed bacterial cultures and by comparing the results with those obtained by conventional qPCR and amplicon sequencing methods. Quantitative results obtained by the NiCE chip were comparable to those obtained by conventional qPCR. In addition, the NiCE chip was successfully applied to examine the abundance and diversity of N cycle functional genes in wastewater samples. Although nonspecific amplification was detected on the NiCE chip, this can be overcome by optimizing the primer sequences in the future. As the NiCE chip can provide a high-throughput format to quantify and prepare sequence libraries for multiple N cycle functional genes, this tool should advance our ability to explore N cycling in various samples.IMPORTANCE We report a novel approach, namely, the nitrogen cycle evaluation (NiCE) chip, by using microfluidic qPCR chip technology. By sequencing the amplicons recovered from the NiCE chip, we can assess the diversities of N cycle functional genes. The NiCE chip technology is applicable to analysis of the temporal dynamics of N cycle gene transcription in wastewater treatment bioreactors. The NiCE chip can provide a high-throughput format to quantify and prepare sequence libraries for multiple N cycle functional genes. While there is room for future improvement, this tool should significantly advance our ability to explore the N cycle in various environmental samples.

SUBMITTER: Oshiki M

PROVIDER: S-EPMC5881049 | biostudies-literature | 2018 Apr

REPOSITORIES: biostudies-literature

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Nitrogen Cycle Evaluation (NiCE) Chip for Simultaneous Analysis of Multiple N Cycle-Associated Genes.

Oshiki Mamoru M Segawa Takahiro T Ishii Satoshi S

Applied and environmental microbiology 20180402 8

Various microorganisms play key roles in the nitrogen (N) cycle. Quantitative PCR (qPCR) and PCR amplicon sequencing of N cycle functional genes allow us to analyze the abundance and diversity of microbes responsible for N-transforming reactions in various environmental samples. However, analysis of multiple target genes can be cumbersome and expensive. PCR-independent analysis, such as metagenomics and metatranscriptomics, is useful but expensive, especially when we analyze multiple samples and ...[more]

PMID: 29427421

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Project description:Biophysical properties are widely used to detect pathophysiological processes of vascular diseases or clinical states. For early detection of cardiovascular diseases, it is necessary to simultaneously measure multiple biophysical properties in a microfluidic environment. However, a microfluidic-based technique for measuring multiple biophysical properties has not been demonstrated. In this study, a simple measurement method was suggested to quantify three biophysical properties of blood, including red blood cell (RBC) deformability, RBC aggregation, and hematocrit. To demonstrate the suggested method, a microfluidic device was constructed, being composed of a big-sized channel (BC), a parallel micropillar (MP), a main channel, a branch channel, inlet, and outlets. By operating a single syringe pump, blood was supplied into the inlet of the microfluidic device, at a periodic on-off profile (i.e., period = 240 s). The RBC deformability index (DI) was obtained by analyzing the averaged blood velocity in the branch channel. Additionally, the RBC aggregation index (AIN) and the hematocrit index (HiBC) were measured by analyzing the image intensity of blood flows in the MP and the BC, respectively. The corresponding contributions of three influencing factors, including the turn-on time (Ton), the amplitude of blood flow rate (Q₀), and the hematocrit (Hct) on the biophysical indices (DI, AIN, and HiBC) were evaluated quantitatively. As the three biophysical indices varied significantly with respect to the three factors, the following conditions (i.e., Ton = 210 s, Q₀ = 1 mL/h, and Hct = 50%) were maintained for consistent measurement of biophysical properties. The proposed method was employed to detect variations of biophysical properties depending on the concentrations of autologous plasma, homogeneous hardened RBCs, and heterogeneous hardened RBCs. Based on the observations, the proposed method exhibited significant differences in biophysical properties depending on base solutions, homogeneous hardened RBCs (i.e., all RBCs fixed with the same concentration of glutaraldehyde solution), and heterogeneous hardened RBCs (i.e., partially mixed with normal RBCs and homogeneous hardened RBCs). Additionally, the suggested indices (i.e., DI, AIN, and HiBC) were effectively employed to quantify three biophysical properties, including RBC deformability, RBC aggregation, and hematocrit.

Dataset Information

Nitrogen Cycle Evaluation (NiCE) Chip for Simultaneous Analysis of Multiple N Cycle-Associated Genes.

Publications

Nitrogen Cycle Evaluation (NiCE) Chip for Simultaneous Analysis of Multiple N Cycle-Associated Genes.

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