A new mathematical model for relative quantification in real-time RT-PCR

Pfaffl, Michael W.

doi:10.1093/nar/29.9.e45

Cited by 29,772 publications

(18,857 citation statements)

References 19 publications

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“…Relative amounts of DNA at the repair locus in different samples were normalized against the ARO1 locus on chr.IVR (OSM1006 + OSM1007). The fraction of non‐homologous DNA ends remaining was quantified relative to the −1 h time point (prior to DSB induction) using the efficiency‐corrected comparative quantitation method (∆∆ C t ) (Pfaffl, 2001). The fraction of DNA ends cleaved after SSA was calculated as [1—fraction of DNA ends remaining ].…”

Section: Methodsmentioning

confidence: 99%

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Unloading of homologous recombination factors is required for restoring double‐stranded DNA at damage repair loci

et al. 2017

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Cells use homology‐dependent DNA repair to mend chromosome breaks and restore broken replication forks, thereby ensuring genome stability and cell survival. DNA break repair via homology‐based mechanisms involves nuclease‐dependent DNA end resection, which generates long tracts of single‐stranded DNA required for checkpoint activation and loading of homologous recombination proteins Rad52/51/55/57. While recruitment of the homologous recombination machinery is well characterized, it is not known how its presence at repair loci is coordinated with downstream re‐synthesis of resected DNA. We show that Rad51 inhibits recruitment of proliferating cell nuclear antigen (PCNA), the platform for assembly of the DNA replication machinery, and that unloading of Rad51 by Srs2 helicase is required for efficient PCNA loading and restoration of resected DNA. As a result, srs2Δ mutants are deficient in DNA repair correlating with extensive DNA processing, but this defect in srs2Δ mutants can be suppressed by inactivation of the resection nuclease Exo1. We propose a model in which during re‐synthesis of resected DNA, the replication machinery must catch up with the preceding processing nucleases, in order to close the single‐stranded gap and terminate further resection.

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Section: Methodsmentioning

confidence: 99%

“…Relative DNA amounts in different samples were normalized to the ARO1 locus on chr.IVR (OSM1006 + OSM1007). De novo telomere addition was quantified relative to 0 h (prior to DSB induction) using efficiency‐corrected comparative quantitation method (∆∆ C t ) (Pfaffl, 2001). …”

Section: Methodsmentioning

confidence: 99%

Unloading of homologous recombination factors is required for restoring double‐stranded DNA at damage repair loci

et al. 2017

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“…Gene expression data were analysed using the REST 2009 software [21] and the provided p value used for the likelihood of up-regulation or down-regulation of each gene between treated and control groups.…”

Section: Methodsmentioning

confidence: 99%

Lentinula edodes β-glucan enriched diet induces pro- and anti-inflammatory macrophages in rabbit

Crespo

Guillén²,

Pablo-Maiso

et al. 2017

Food & Nutrition Research

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ABSTRACTβ-glucans exhibited in cell walls of several pathogens as bacteria or fungi are sensed by pathogen recognition receptors such as scavenger receptors present in antigen presenting cells, i.e., macrophages. β-glucans obtained from Shiitake mushrooms were chemically characterized. A β-glucan supplemented diet was assayed for 30 days in rabbits aiming to characterize the immune response elicited in blood-derived macrophages. M1 and M2 profiles of macrophage differentiation were confirmed in rabbits by in vitro stimulation with IFN-γ and IL-4 and marker quantification of each differentiation pathway. Blood derived macrophages from rabbits administered in vivo with the β-glucan supplemented diet showed higher IL-4, IFN-γ and RAGE together with lower IL-10 relative expression, indicative of an ongoing immune response. Differences in IL-1β, IL-13 and IL-4 expression were also found in rabbit sera by ELISA suggesting further stimulation of the adaptive response. Recent challenges in the rabbit industry include the search of diet supplements able to elicit an immune stimulation with particular interest in facing pathogens such as viruses or bacteria. β–glucans from fungi may contribute to maintain an immune steady state favouring protection and thus reducing antibiotic treatment.

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“…For stimulated spleen cells, gene expression ratios were calculated using the Pfaffl method [Pfaffl, 2001]. For genes in the HPA axis, the gene expression was determined relative to the housekeeping gene.…”

Section: Methodsmentioning

confidence: 99%

A short‐term extremely low frequency electromagnetic field exposure increases circulating leukocyte numbers and affects HPA‐axis signaling in mice

Kleijn

Ferwerda

Wiese

et al. 2016

Bioelectromagnetics

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There is still uncertainty whether extremely low frequency electromagnetic fields (ELF‐EMF) can induce health effects like immunomodulation. Despite evidence obtained in vitro, an unambiguous association has not yet been established in vivo. Here, mice were exposed to ELF‐EMF for 1, 4, and 24 h/day in a short‐term (1 week) and long‐term (15 weeks) set‐up to investigate whole body effects on the level of stress regulation and immune response. ELF‐EMF signal contained multiple frequencies (20–5000 Hz) and a magnetic flux density of 10 μT. After exposure, blood was analyzed for leukocyte numbers (short‐term and long‐term) and adrenocorticotropic hormone concentration (short‐term only). Furthermore, in the short‐term experiment, stress‐related parameters, corticotropin‐releasing hormone, proopiomelanocortin (POMC) and CYP11A1 gene‐expression, respectively, were determined in the hypothalamic paraventricular nucleus, pituitary, and adrenal glands. In the short‐term but not long‐term experiment, leukocyte counts were significantly higher in the 24 h‐exposed group compared with controls, mainly represented by increased neutrophils and CD4 ± lymphocytes. POMC expression and plasma adrenocorticotropic hormone were significantly lower compared with unexposed control mice. In conclusion, short‐term ELF‐EMF exposure may affect hypothalamic‐pituitary‐adrenal axis activation in mice. Changes in stress hormone release may explain changes in circulating leukocyte numbers and composition. Bioelectromagnetics. 37:433–443, 2016. © 2016 The Authors. Bioelectromagnetics Published by Wiley Periodicals, Inc.

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A new mathematical model for relative quantification in real-time RT-PCR

Cited by 29,772 publications

References 19 publications

Unloading of homologous recombination factors is required for restoring double‐stranded DNA at damage repair loci

Unloading of homologous recombination factors is required for restoring double‐stranded DNA at damage repair loci

Lentinula edodes β-glucan enriched diet induces pro- and anti-inflammatory macrophages in rabbit

A short‐term extremely low frequency electromagnetic field exposure increases circulating leukocyte numbers and affects HPA‐axis signaling in mice

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