Experimental validation of novel and conventional approaches to quantitative real-time PCR data analysis

Department of Integrative and Molecular Neuroscience, Division of Neuroscience and Psychological Medicine, Faculty of Medicine, Imperial College London, Charing Cross Hospital, Fulham Palace Road, London W6 8RF, UK.
Nucleic Acids Research (Impact Factor: 9.11). 08/2003; 31(14):e73.
Source: PubMed


Real-time PCR is being used increasingly as the method of choice for mRNA quantification, allowing rapid analysis of gene expression from low quantities of starting template. Despite a wide range of approaches, the same principles underlie all data analysis, with standard approaches broadly classified as either absolute or relative. In this study we use a variety of absolute and relative approaches of data analysis to investigate nocturnal c-fos expression in wild-type and retinally degenerate mice. In addition, we apply a simple algorithm to calculate the amplification efficiency of every sample from its amplification profile. We confirm that nocturnal c-fos expression in the rodent eye originates from the photoreceptor layer, with around a 5-fold reduction in nocturnal c-fos expression in mice lacking rods and cones. Furthermore, we illustrate that differences in the results obtained from absolute and relative approaches are underpinned by differences in the calculated PCR efficiency. By calculating the amplification efficiency from the samples under analysis, comparable results may be obtained without the need for standard curves. We have automated this method to provide a means of streamlining the real-time PCR process, enabling analysis of experimental samples based upon their own reaction kinetics rather than those of artificial standards.

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    • "Data analysis for real-time PCR (DART-PCR) (Peirson et al., 2003) was used for analysing the raw qPCR data. For every sample, DART- PCR enables calculation of threshold cycles and amplification efficiencies. "
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    ABSTRACT: The molecular mechanisms activated by environmental contaminants and natural stressors such as freezing need to be investigated in order to better understand the mechanisms of interaction and potential effects that combined stressors may have on organisms. Using the freeze tolerant earthworm Dendrobaena octaedra as model species, we exposed worms to freezing and exposure to sublethal copper in a factorial design and investigated the transcription of candidate genes for metal and cold stress. We hypothesized that both freezing and copper would induce transcription of genes coding for heat shock proteins (hsp10 and hsp70), metallothioneins (mt1 and mt2) and glutathione-S-transferase (gst), and that the combined effects of these two stressors would be additive. The gene transcripts hsp10, hsp70 and gst were significantly upregulated by freezing, but only hsp10 was upregulated by copper. We found that copper at the time of sampling had no effect on transcription of two metallothionein genes whereas transcription was strongly upregulated by freezing. Moreover there was a significant interaction causing more than additive transcription rates of mt1 in the copper/freezing treatment suggesting that freeze-induced cellular dehydration increases the concentration of free copper ions in the cytosol. This metallothionein response to freezing is likely adaptive and possibly provides protection against freeze-induced elevated metal concentrations in the cytosol and excess ROS levels due to hypoxia during freezing. Copyright © 2015. Published by Elsevier Inc.
    Comparative Biochemistry and Physiology Part C Toxicology & Pharmacology 08/2015; DOI:10.1016/j.cbpc.2015.08.008 · 2.30 Impact Factor
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    • "The signal was monitored using ABI Prism 7500 Real-time PCR System (Life technologies, Carlsbad, CA, USA). Standard curve method was used for relative quantification of target genes (Peirson et al., 2003). The rpsJ and dnaN genes were used as a single copy control in all experiments. "
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    ABSTRACT: GI-VII-6 is a chromosomally integrated multidrug resistance genomic island harbored by a specific clone of Salmonella enterica serovar Typhimurium (S.Typhimurium). It contains a gene encoding CMY-2 β-lactamase (bla CMY-2), and therefore contributes to extended-spectrum cephalosporin resistance. To elucidate the significance of GI-VII-6 on adaptive evolution, spontaneous mutants of S. Typhimurium strain L-3553 were selected on plates containing cefotaxime (CTX). The concentrations of CTX were higher than its minimum inhibition concentration to the parent strain. The mutants appeared on the plates containing 12.5 and 25 mg/L CTX at a frequency of 10(-6) and 10(-8), respectively. No colonies were observed at higher CTX concentrations. The copy number of bla CMY-2 increased up to 85 per genome in the mutants, while the parent strain contains one copy of that in the chromosome. This elevation was accompanied by increased amount of transcription. The bla CMY-2 copy number in the mutants drastically decreased in the absence of antimicrobial selection pressure. Southern hybridization analysis and short-read mapping indicated that the entire 125 kb GI-VII-6 or parts of it were tandemly amplified. GI-VII-6 amplification occurred at its original position, although it also transposed to other locations in the genome in some mutants, including an endogenous plasmid in some of the mutants, leading to the amplification of GI-VII-6 at different loci. Insertion sequences were observed at the junction of the amplified regions in the mutants, suggesting their significant roles in the transposition and amplification. Plasmid copy number in the selected mutants was 1.4 to 4.4 times higher than that of the parent strain. These data suggest that transposition and amplification of the bla CMY-2-containing region, along with the copy number variation of the plasmid, contributed to the extensive amplification of bla CMY-2 and increased resistance to CTX.
    Frontiers in Microbiology 02/2015; 6:78. DOI:10.3389/fmicb.2015.00078 · 3.99 Impact Factor
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    • "To date, there are four cases in which the genetic basis of white-fl owered morphs has been identifi ed, and these involve an array of mutations affecting both coding and cis -regulatory regions of structural genes and transcription factors. The white morph of the arctic mustard Parrya nudicaulis is due to a cis -regulatory mutation in Chs ( Dick et al., 2011 ), and a transposon insertion in the coding region of this gene causes white fl owers in Ipomoea purpurea ( Habu et al., 1998 ; Coberly and Rausher, 2003 ). White morphs of I. purpurea have also been associated with a loss-of-function allele (containing two deletions) at the Ipmyb1 locus that encodes an R2R3-MYB transcription factor ( Chang et al., 2005 ). "
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    ABSTRACT: • White forms of typically pigmented flowers are one of the most common polymorphisms in flowering plants. Although the range of genetic changes that give rise to white phenotypes is well known from model systems, few studies have identified causative mutations in natural populations.• Here we combine genetic studies, in vitro enzyme assays, and biochemical analyses to identify the mechanism underlying the loss of anthocyanin pigment production in the naturally occurring white-flowered morph of Iochroma calycinum (Solanaceae).• Comparison of anthocyanin gene sequences revealed a putative loss-of-function mutation, an 11 amino-acid deletion in dihydroflavonol 4-reductase (DFR), in the white morph. Functional assays of Dfr alleles from blue and white morphs demonstrated that this deletion results in a loss of enzymatic activity, indicating that the deletion could be solely responsible for the lack of pigment production. Consistent with this hypothesis, quantitative PCR showed no significant differences in expression of anthocyanin genes between the morphs. Also, thin layer chromatography confirmed that the white morph continues to accumulate compounds upstream of the DFR enzyme.• Collectively, these experiments indicate that the structural mutation at Dfr underlies the rare white flower morph of I. calycinum. This study is one of only a few examples where a flower color polymorphism is due to a loss-of-function mutation in the coding region of an anthocyanin enzyme. The rarity of such mutations in nature suggests that negative consequences prevent fixation across populations. © 2015 Botanical Society of America, Inc.
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