ProbeLibrary: A new method for faster design and execution of quantitative real-time PCR

Mouritzen, Peter; Noerholm, Mikkel; Nielsen, P. H.; Jacobsen, Nana; Lomholt, Christian; Pfundheller, Henrik M.; Tolstrup, Niels

doi:10.1038/nmeth0405-313

Cited by 33 publications

(20 citation statements)

References 2 publications

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“…The primers and probes were designed using the Universal ProbeLibrary (UPL) Assay Design Center (Roche). The UPL probes were prevalidated short TaqMan probes that contained locked nucleic acids, which were labeled with 6-carboxyfluorescein (6-FAM) at the 5= end and dark quencher near the 3= end (34). The nucleotide sequences of the target genes were obtained from GenBank and aligned using CLUSTALW (35).…”

Section: Methodsmentioning

confidence: 99%

Simultaneous Quantification of Multiple Food- and Waterborne Pathogens by Use of Microfluidic Quantitative PCR

Ishii

Segawa

Okabe

2013

Appl Environ Microbiol

127

159

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bThe direct quantification of multiple pathogens has been desired for diagnostic and public health purposes for a long time. In this study, we applied microfluidic quantitative PCR (qPCR) technology to the simultaneous detection and quantification of multiple food-and waterborne pathogens. In this system, multiple singleplex qPCR assays were run under identical detection conditions in nanoliter-volume chambers that are present in high densities on a chip. First, we developed 18 TaqMan qPCR assays that could be run in the same PCR conditions by using prevalidated TaqMan probes. Specific and sensitive quantification was achieved by using these qPCR assays. With the addition of two previously validated TaqMan qPCR assays, we used 20 qPCR assays targeting 10 enteric pathogens, a fecal indicator bacterium (general Escherichia coli), and a process control strain in the microfluidic qPCR system. We preamplified the template DNA to increase the sensitivity of the qPCR assays. Our results suggested that preamplification was effective for quantifying small amounts of the template DNA without any major impact on the sensitivity, efficiency, and quantitative performance of qPCR. This microfluidic qPCR system allowed us to detect and quantify multiple pathogens from fecal samples and environmental water samples spiked with pathogens at levels as low as 100 cells/liter. These results suggest that the routine monitoring of multiple pathogens in food and water samples is now technically feasible. This method may provide more reliable information for risk assessment than the current fecal contamination indicator approach.

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Section: Methodsmentioning

confidence: 99%

Simultaneous Quantification of Multiple Food- and Waterborne Pathogens by Use of Microfluidic Quantitative PCR

Ishii

Segawa

Okabe

2013

Appl Environ Microbiol

127

159

View full text Add to dashboard Cite

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“…Labelled probes were purchased from the Exiqon Human Universal Probe Library system [30] (Roche, Switzerland), with amplification primers obtained from MWG (Germany). RNA was extracted from MCF7 and MCF10a cell lines using RNeasy reagents (Qiagen, Germany) and reverse transcribed to cDNA using Taqman Reverse transcription reagents (Applied Biosystems, USA) with random hexamers as primers.…”

Section: Methodsmentioning

confidence: 99%

The utility of MAS5 expression summary and detection call algorithms

et al. 2007

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“…RNA was extracted from 1st and 2nd pass cells using RNeasy mini kit (Qiagen, GmbH, Germany), and cDNA obtained by MuLV Reverse Transcriptase (Applied Biosystems, CA, USA). 1 lg of cDNA was amplified with Light Cycler and Universal ProbeLibrary system (Roche Applied Science, Italy) [12].…”

Section: Gene Expressionmentioning

confidence: 99%

Ex vivo observation of human intervertebral disc tissue and cells isolated from degenerated intervertebral discs

et al. 2012

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Purpose Disc degeneration, and associated low back pain, are a primary cause of disability. Disc degeneration is characterized by dysfunctional cells and loss of proteoglycans: since intervertebral tissue has a limited capacity to regenerate, this process is at present considered irreversible. Recently, cell therapy has been suggested to provide more successful treatment of IVD degeneration. To understand the potential of cells to restore IVD structure/function, tissue samples from degenerated IVD versus healthy discs have been compared. Methods Discal tissue from 27 patients (40.17 ± 11 years) undergoing surgery for degenerative disc disease (DDD), DDD ? herniation and congenital scoliosis, as controls, was investigated. Cells and matrix in the nucleus pulposus (NP) and annulus fibrosus (AF) were characterized by histology.AF-and NP-derived cells were isolated, expanded and characterized for senescence and gene expression. Three-dimensional NP pellets were cultured and stained for glycosaminoglycan formation.Results Phenotypical markers of degeneration, such as cell clusters, chondrons, and collagen disorganization were seen in the degenerate samples. In severe degeneration, granulation tissue and peripheral vascularization were observed. No correlation was found between the Pfirrmann clinical score and the extent of degeneration. Conclusion The tissue disorganization in degenerate discs and the paucity of cells out of cluster/chondron association, make the IVD-derived cells an unreliable option for disc regeneration.

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ProbeLibrary: A new method for faster design and execution of quantitative real-time PCR

Cited by 33 publications

References 2 publications

Simultaneous Quantification of Multiple Food- and Waterborne Pathogens by Use of Microfluidic Quantitative PCR

Simultaneous Quantification of Multiple Food- and Waterborne Pathogens by Use of Microfluidic Quantitative PCR

The utility of MAS5 expression summary and detection call algorithms

Ex vivo observation of human intervertebral disc tissue and cells isolated from degenerated intervertebral discs

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