Assumption-free analysis of quantitative real-time polymerase chain reaction (PCR) data

Ramakers, Christian; Ruijter, Jan M.; Deprez, Ronald H. Lekanne; Moorman, Antoon F.M.

doi:10.1016/s0304-3940(02)01423-4

Cited by 3,309 publications

(2,573 citation statements)

References 7 publications

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“…PCR amplification efficiencies were measured on individual amplification plots using LinReg PCR. 44 The software REST 384 (http://www. gene-quantification.info/) 45 was used to analyze the relative expression of our measured transcripts using an efficiency corrected ratio.…”

Section: Quantitative Real-time Rt-pcrmentioning

confidence: 99%

Incremental expression of Tlr4 correlates with mouse resistance to Salmonella infection and fine regulation of relevant immune genes

Larivière

Wilkinson

et al. 2006

Genes Immun

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Section: Quantitative Real-time Rt-pcrmentioning

confidence: 99%

Incremental expression of Tlr4 correlates with mouse resistance to Salmonella infection and fine regulation of relevant immune genes

Larivière

Wilkinson

et al. 2006

Genes Immun

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“…For accuracy of the analysis, the mean primer efficiency was estimated using the linear phase of all individual reaction amplification curves (Ramakers et al . 2003), calculated using the LinRegPCR package (Tuomi et al . 2010).…”

Section: Methodsmentioning

confidence: 99%

Overexpression of a NAC transcription factor delays leaf senescence and increases grain nitrogen concentration in wheat

et al. 2015

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Increasing the duration of leaf photosynthesis during grain filling using slow‐senescing functional stay‐green phenotypes is a possible route for increasing grain yields in wheat (Triticum aestivum L.). However, delayed senescence may negatively affect nutrient remobilisation and hence reduce grain protein concentrations and grain quality. A novel NAC1‐type transcription factor (hereafter TaNAC‐S) was identified in wheat, with gene expression located primarily in leaf/sheath tissues, which decreased during post‐anthesis leaf senescence. Expression of TaNAC‐S in the second leaf correlated with delayed senescence in two doubled‐haploid lines of an Avalon × Cadenza population (lines 112 and 181), which were distinct for leaf senescence. Transgenic wheat plants overexpressing TaNAC‐S resulted in delayed leaf senescence (stay‐green phenotype). Grain yield, aboveground biomass, harvest index and total grain N content were unaffected, but NAC over‐expressing lines had higher grain N concentrations at similar grain yields compared to non‐transgenic controls. These results indicate that TaNAC‐S is a negative regulator of leaf senescence, and that delayed leaf senescence may lead not only to increased grain yields but also to increased grain protein concentrations.

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“…16 We estimated the efficiency for each single sample tube using the slope of the exponential phase as described by Ramakers et al 17 Furthermore, as expected, controls for unmethylated DNA (a purified native amplicon) and fully methylated DNA (the same amplicon treated with the DNA methylase M.SssI (New England Biolabs, Ipswich, MA) yielded 0% and 100% DNA methylation patterns.…”

Section: Methodsmentioning

confidence: 99%

Epigenetic regulation of insulin resistance in nonalcoholic fatty liver disease: Impact of liver methylation of the peroxisome proliferator-activated receptor γ coactivator 1α promoter

et al. 2010

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Insulin resistance (IR) and mitochondrial dysfunction play a central role in the pathophysiology of nonalcoholic fatty liver disease (NAFLD). We hypothesized that genetic factors and epigenetic modifications occurring in the liver contribute to the IR phenotype. We specifically examined whether fatty liver and IR are modified by hepatic DNA methylation of the peroxisome proliferator-activated receptor c coactivator 1a (PPARGC1A) and mitochondrial transcription factor A (TFAM) promoters, and also evaluated whether liver mitochondrial DNA (mtDNA) content is associated with NAFLD and IR. We studied liver biopsies obtained from NAFLD patients in a case-control design. After bisulfite treatment of DNA, we used methylation-specific polymerase chain reaction (PCR) to assess the putative methylation of three CpG in the PPARGC1A and TFAM promoters. Liver mtDNA quantification using nuclear DNA (nDNA) as a reference was evaluated by way of realtime PCR. Liver PPARGC1A methylated DNA/unmethylated DNA ratio correlated with plasma fasting insulin levels and homeostasis model assessment of insulin resistance (HOMA-IR); TFAM methylated DNA/unmethylated DNA ratio was inversely correlated with insulin levels. PPARGC1A promoter methylation was inversely correlated with the abundance of liver PPARGC1A messenger RNA. The liver mtDNA/nDNA ratio was significantly higher in control livers compared with NAFLD livers. mtDNA/nDNA ratio was inversely correlated with HOMA-IR, fasting glucose, and insulin and was inversely correlated with PPARGC1A promoter methylation. Conclusion: Our data suggest that the IR phenotype and the liver transcriptional activity of PPARGC1A show a tight interaction, probably through epigenetic modifications. Decreased liver mtDNA content concomitantly contributes to peripheral IR.

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Assumption-free analysis of quantitative real-time polymerase chain reaction (PCR) data

Cited by 3,309 publications

References 7 publications

Incremental expression of Tlr4 correlates with mouse resistance to Salmonella infection and fine regulation of relevant immune genes

Incremental expression of Tlr4 correlates with mouse resistance to Salmonella infection and fine regulation of relevant immune genes

Overexpression of a NAC transcription factor delays leaf senescence and increases grain nitrogen concentration in wheat

Epigenetic regulation of insulin resistance in nonalcoholic fatty liver disease: Impact of liver methylation of the peroxisome proliferator-activated receptor γ coactivator 1α promoter

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