Papers by Keyword: Real Time-PCR

Abstract: In this paper, we design a temperature sensing system based on a DSP chip TMS320F2812 for a real-time PCR instrument. In this system, we design a 4-wire temperature sensing module with a thermal resistor PT100 to measure the temperature and convert it into a voltage signal. Then the voltage signal is digitalized by the ADC module. At last, the data are transferred to a PC through the serial port. The experimental results show that the linearity of the system is 0.99 with the standard deviation of 0.0419.

Abstract: Trehalose (α-D-glucopyranosyl-1,1-α-D-glucopyranoside) is a non-reducing disaccharide. It is currently thought that just trace level of trehalose was detected in plants, and that trehalose metabolic pathway was significantly related to stress tolerance. In this study, we report that expression levels of three genes with regard to trehalose metabolic pathway were measured in Arabidopsis thaliana, including AtTPS1, AtTPPA and AtTRE1. As a result, transcriptional levels of these genes are the highest in floral organ, and the expression of AtTRE1 is much more than AtTPS1 and AtTPPA. Additionally, we present transcriptional response analyses in drought and heat stresses, which have shown the changes of these genes expression from tolerance in early stress to senescence in later stress.

Abstract: The geomicrobiology of sulfidic mine dumps is reviewed. More than 30 microbiological studies of sulfidic mine dumps have been published. Mainly culturing approaches such as most probable number (MPN) or agar plates were used to study the microbial communities. More recently, molecular biological techniques such as FISH, CARD-FISH, Q-PCR, T-RFLP, DGGE, or cloning have been applied to quantify microorganisms and to investigate the microbial diversity. Aerobic Fe(II)- and sulfur compound oxidizing microorganisms oxidize pyrite, pyrrhotite and other metal sulfides and play an important role in the formation of acid mine drainage (AMD). Anaerobic microorganisms such as Fe(III)-reducing microorganisms dissolve Fe(III)(hydr)oxides and may thereby release adsorbed or precipitated metals. Sulfate-reducing microorganisms precipitate and immobilize metals. In addition to the microbial communities several biogeochemical processes have been analyzed in mine dumps. Pyrite or pyrrhotite oxidation rates have been measured by different techniques: Column experiments, humidity cells, microcalorimetry, or oxygen consumption measurements. Analyses of stable isotopes of iron, oxygen and sulfur have yielded valuable information on biogeochemical reactions. The microbiology and the biogeochemical processes in sulfidic mine dumps have to be understood for control and prevention of AMD generation and to provide different possibilities for remediation concepts. Today, remediation measures, e.g. under water storage of the waste or covering of the dumps, focus on the inhibition of pyrite oxidation to keep the toxic compounds inside the mine waste dumps. As an alternative to the inhibition of pyrite oxidation, metals which also have economic value could be extracted from mine dumps by the application of different metal extraction technologies including bioleaching.

Abstract: Microbial heap bioleaching is being used as an industrial process to recover copper from low grade ores. It is known that a consortium of different microorganisms participates in this process. Therefore identification and quantification of communities inhabiting heap bioleaching operations is a key step for understanding the dynamics and role of these microorganisms in the process. A quantitative real-time PCR approach was used to investigate the microbial dynamics in this process. To study the microbial population inhabiting a low-grade copper sulphide ore bioleaching industrial heap process at Escondida Mine in Chile, 16S rRNA genetic libraries were constructed using bacterial and archaeal universal primers. Phylogenetic analyses of sequences retrieved from genetic libraries showed that the community is mainly composed by microoganisms related to Acidithiobacillus ferrooxidans (2 strains), Acidithiobacillus thiooxidans, Leptospirillum ferrooxidans, Leptospirillum ferriphilum and the archaea Ferroplasma. Specific primers for real-time PCR determination were designed and tested to amplify each of the sequences obtained by cloning. Standard curves for real time PCR were performed using plasmid DNA from selected clones. This methodology is actually being used to monitor relevant microorganisms inhabiting this low-grade copper sulphide ore bioleaching industrial heap.

Abstract: Cemented layers predominantly consisting of gels/poorly crystalline mineral phases have been formed as a consequence of mineral weathering in sulfidic tailings near Freiberg, Saxony, Germany. These layers function as natural attenuation barrier for toxic compounds and reduce oxidation and erosion processes of tailings surfaces. Quantitative molecular biological and cultivation methods were applied to investigate the role of microorganisms for mineral weathering and cemented layer formation. High resolution depth profiles of numbers of microorganisms showed maximal cell numbers in the oxidation zone where cemented layers had been formed. Highest total cell numbers of >109 cells g-1 dry weight (dw) were detected by SybrGreen direct counting. Using quantitative real-time PCR (Q-PCR) between 107 and 109 Bacteria g-1 dw and up to 108 Archaea g-1 dw were determined. As well high numbers of cultivable and living Bacteria could be detected by MPN (most probable number) for Fe(II)- and S-oxidizers and CARD-FISH (catalyzed reporter deposition - fluorescence in situ hybridization). Overall, the high numbers of microorganisms determined with various quantification techniques argue for a significant role of microorganisms in cemented layer formation due to microbial mineral weathering. It is hypothesized that EPS (extracellular polymeric substances) mediate the formation of secondary mineral phases.

Abstract: Three-dimensional gelatin-chondroitin 6 sulphate-hyanuronic acid biomatrix was used as the scaffold to investigate the phenotypic and molecular expression in human keratinocytes (K) and dermal fibroblasts (FB) in three different culture conditions in vitro. The cells were cultured in either monolayer (K or FB only) or coculture (K&FB) model. The deposition of basement membrane proteins secreted by these two kinds of cells was quantitatively characterized by real-time PCR. In the results, dermal fibroblasts were shown to synthesize and deposit laminin 5, type IV and type VII collagen, whereas keratinocytes produced integrin alpha 6 and beta 4 as well as laminin 5 and collagen type IV, VII. Interestingly, the integrin beta 4 subunit was not expressed either in keratinocytes or dermal fibroblasts monoculture but was seen in organotypic coculture model in the early culture period. Furthermore, we found that the expression of those marker compounds was reciprocally regulated when keratinocytes and dermal fibroblasts were cultured together. These results indicated that keratinocyes and dermal fibroblasts worked together to reconstruct dermal-epidermal basement membrane (BM) zone. In brief, our data provide the first time in directly quantifying the expression of BM proteins by using real-time PCR, and also demonstrate that BM proteins were regulated by cell-cell interaction.