Amplification efficiency: linking baseline and bias in the analysis of quantitative PCR data. Nucleic Acids Res 37(6):e45

Heart Failure Research Center, Academic Medical Center, University of Amsterdam, The Netherlands.
Nucleic Acids Research (Impact Factor: 9.11). 03/2009; 37(6):e45. DOI: 10.1093/nar/gkp045
Source: PubMed

ABSTRACT Despite the central role of quantitative PCR (qPCR) in the quantification of mRNA transcripts, most analyses of qPCR data
are still delegated to the software that comes with the qPCR apparatus. This is especially true for the handling of the fluorescence
baseline. This article shows that baseline estimation errors are directly reflected in the observed PCR efficiency values
and are thus propagated exponentially in the estimated starting concentrations as well as ‘fold-difference’ results. Because
of the unknown origin and kinetics of the baseline fluorescence, the fluorescence values monitored in the initial cycles of
the PCR reaction cannot be used to estimate a useful baseline value. An algorithm that estimates the baseline by reconstructing
the log-linear phase downward from the early plateau phase of the PCR reaction was developed and shown to lead to very reproducible
PCR efficiency values. PCR efficiency values were determined per sample by fitting a regression line to a subset of data points
in the log-linear phase. The variability, as well as the bias, in qPCR results was significantly reduced when the mean of
these PCR efficiencies per amplicon was used in the calculation of an estimate of the starting concentration per sample.

Download full-text


Available from: Maurice J B van den Hoff, Sep 27, 2015
1 Follower
61 Reads
  • Source
    • "h sample was examined in three technical replicates , and dissociation curves were analyzed to verify the specificity of each reaction . Cycle threshold ( Ct ) values were extracted with the Light Cycler 480 SV1 . 5 . 0 software ( Roche ) using the second derivative calculation and LinReg 11 . 1 software was used to calculate reaction efficiency ( Ruijter et al . , 2009 ) . The relative expression of each gene was obtained according to the Pfaffl equation ( Pfaffl , 2001 ) , using leaves of control plants at 0 dpi as the calibrator . The γ - chain elongation factor 1 gene ( EF1 - γ ) was used as reference gene with constitutive expression for data normalization ( Dufour et al . , 2013 ) . For each samp"
    [Show abstract] [Hide abstract]
    ABSTRACT: Powdery mildew caused by Erysiphe necator is one of the most important grapevine diseases in several viticulture areas, and high fungicide input is required to control it. However, numerous synthetic chemical pesticides are under scrutiny due to concerns about their impact on human health and the environment. Biopesticides, such as biogenic elicitors, are a promising alternative to chemical fungicides. Although several studies have reported on effective elicitors against grapevine diseases, their efficacy under field conditions has not been investigated extensively or has occurred at rather limited levels. Our goal was to examine the efficacy of a protein-based composition, namely nutrient broth (NB), against powdery mildew under field conditions and to characterize its mechanism of action. Weekly treatments with NB was highly effective in controlling powdery mildew on grapevine across seasons with different disease pressures. The level of disease control achieved with NB was comparable to standard fungicide treatments both on leaves and bunches across three different years. NB has no direct toxic effect on the germination of E. necator conidia, and it activates plant resistance with both systemic and translaminar effect in experiments with artificial inoculation under controlled conditions. NB induced the expression of defense-related genes in grapevine, demonstrating stimulation of plant defense mechanisms, prior to and in the early stages of pathogen infection. NB is a natural derivative from meat and yeast, substances that tend not to raise concerns about toxicological and ecotoxicological properties. NB represents a valid control tool for integrated plant protection programs against powdery mildew, to reduce the use of synthetic pesticides on grapevine.
    Frontiers in Plant Science 09/2015; 6:715. DOI:10.3389/fpls.2015.00715 · 3.95 Impact Factor
  • Source
    • "Then, a window of linearity was set, and PCR efficiencies per sample were calculated. With the mean PCR efficiency per amplicon, the quantification cycle value per sample and the fluorescence threshold set to determine it, the starting concentration per sample, expressed in arbitrary fluorescence units, was calculated [27] [28]. GAPDH (glyceraldehyde-3-phosphate dehydrogenase) and HPRT1 (hypoxanthine phosphoribosyltransferase 1) were used as housekeeping genes to control for loading errors and to normalize RNA concentration. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Aims: Dysregulation of the transforming growth factor beta (TGF-β) 1 pathway has been associated with either syndromic or isolated mitral valve (MV) prolapse due to myxoid degeneration (floppy MV). The activation of Smad receptor-mediated intracellular TGF-β pathway and its effect on adherens junction (AJ) molecular pattern of activated valvular interstitial cells (VICs) in MV prolapse are herein investigated. Methods: Floppy MV leaflets were obtained from 30 patients (24 males, mean age 55.5±12.7 years) who underwent surgical repair, and 10 age- and sex-matched Homograft Tissue Bank samples served as controls. MV leaflet cellular and extracellular matrix composition, including collagen I and III, was evaluated by histology and transmission electron microscopy. Smad2 active phosphorylated form (p-Smad2), α-smooth muscle actin (α-SMA), and junctional proteins (N-cadherin, cadherin-11, β-catenin, plakoglobin, plakophilin-2) in VICs were assessed by immunohistochemistry and immunofluorescence and confirmed by immunoblotting. Quantitative real-time polymerase chain reaction was carried out for components of TGF-β pathway cascade and filamin A (FLN-A). Results: Floppy MV leaflets were thicker (P<.001) and had higher α-SMA+ cell density (P=.002) and collagen III expression (P<.001) than controls. Enhanced p-Smad2 nuclear immunoreactivity (P<.001) and TGF-β1 gene (P=.045), TIMP1 (P=.020), and CTGF (P=.047) expression but no differences in FLN-A and total Smad2 gene expression levels were found between floppy MV and controls. Higher expression of cadherin-11, either exclusively or in colocalization with N-cadherin, and aberrant presence of plakophilin-2 at the AJ were found in floppy MV vs. Controls: Conclusions: TGF-β1 pathway activation in nonsyndromic MV prolapse induces VICs differentiation into contractile myofibroblasts and is associated with changes in the molecular pattern of the AJ, with increased cadherin-11 and aberrant plakophilin-2 expression. AJ reinforcement might promote latent TGF-β1 activation leading to extracellular matrix remodeling in floppy MV.
    Cardiovascular pathology: the official journal of the Society for Cardiovascular Pathology 09/2015; DOI:10.1016/j.carpath.2015.07.009 · 2.00 Impact Factor
    • "Real-time PCR was performed in duplicates on StepOnePlus Real-time PCR System (Life Technologies, Foster City, USA) using QuantiTect SYBR Green PCR Kit (Qiagen GmbH, Hilden, Germany) in 12.5 l volume with 0.3 M of each primer and 1 l of diluted 1:2 total cDNA (final dilution 1:25) as a template . Abundance of target transcripts was normalized relative to the expression level of the TBP gene, coding a TATA box binding protein, as previously described (Migas et al., 2014) and quantified according to the Ruijter's approach (Ruijter et al., 2009). "
    [Show abstract] [Hide abstract]
    ABSTRACT: The t(8;21) translocation is the most widespread genetic defect found in human acute myeloid leukemia. This translocation results in the RUNX1-RUNX1T1 fusion gene that produces a wide variety of alternative transcripts and influences the course of the disease. The rules of combinatorics and splicing of exons in the RUNX1-RUNX1T1 transcripts are not known. To address this issue, we developed an exon graph model of the fusion gene organization and evaluated its local exon combinatorics by the exon combinatorial index (ECI). Here we show that the local exon combinatorics of the RUNX1-RUNX1T1 gene follows a power-law behavior and i) the vast majority of exons has a low ECI, ii) only a small part is represented by "exons-hubs" of splicing with very high ECI values, and iii) it is scale-free and very sensitive to targeted skipping of "exons-hubs". Stochasticity of the splicing machinery and preferred usage of exons in alternative splicing can explain such behavior of the system. Stochasticity may explain up to 12% of the ECI variance and results in a number of non-coding and unproductive transcripts that can be considered as a noise. Half-life of these transcripts is increased due to the deregulation of some key genes of the nonsense-mediated decay system in leukemia cells. On the other hand, preferred usage of exons may explain up to 75% of the ECI variability. Our analysis revealed a set of splicing-related cis-regulatory motifs that can explain "attractiveness" of exons in alternative splicing but only when they are considered together. Cis-regulatory motifs are guides for splicing trans-factors and we observed a leukemia-specific profile of expression of the splicing genes in t(8;21)-positive blasts. Altogether, our results show that alternative splicing of the RUNX1-RUNX1T1 transcripts follows strict rules and that the power-law component of the fusion gene organization confers a high flexibility to this process. Copyright © 2015. Published by Elsevier Ltd.
    The international journal of biochemistry & cell biology 08/2015; DOI:10.1016/j.biocel.2015.08.017 · 4.05 Impact Factor
Show more