The Real-time Polymerase Chain Reaction

TATAA Biocenter, Medicinargatan 7B, 405 30 Göteborg, Sweden.
Molecular Aspects of Medicine (Impact Factor: 10.24). 04/2006; 27(2-3):95-125. DOI: 10.1016/j.mam.2005.12.007
Source: PubMed


The scientific, medical, and diagnostic communities have been presented the most powerful tool for quantitative nucleic acids analysis: real-time PCR [Bustin, S.A., 2004. A-Z of Quantitative PCR. IUL Press, San Diego, CA]. This new technique is a refinement of the original Polymerase Chain Reaction (PCR) developed by Kary Mullis and coworkers in the mid 80:ies [Saiki, R.K., et al., 1985. Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia, Science 230, 1350], for which Kary Mullis was awarded the 1993 year's Nobel prize in Chemistry. By PCR essentially any nucleic acid sequence present in a complex sample can be amplified in a cyclic process to generate a large number of identical copies that can readily be analyzed. This made it possible, for example, to manipulate DNA for cloning purposes, genetic engineering, and sequencing. But as an analytical technique the original PCR method had some serious limitations. By first amplifying the DNA sequence and then analyzing the product, quantification was exceedingly difficult since the PCR gave rise to essentially the same amount of product independently of the initial amount of DNA template molecules that were present. This limitation was resolved in 1992 by the development of real-time PCR by Higuchi et al. [Higuchi, R., Dollinger, G., Walsh, P.S., Griffith, R., 1992. Simultaneous amplification and detection of specific DNA-sequences. Bio-Technology 10(4), 413-417]. In real-time PCR the amount of product formed is monitored during the course of the reaction by monitoring the fluorescence of dyes or probes introduced into the reaction that is proportional to the amount of product formed, and the number of amplification cycles required to obtain a particular amount of DNA molecules is registered. Assuming a certain amplification efficiency, which typically is close to a doubling of the number of molecules per amplification cycle, it is possible to calculate the number of DNA molecules of the amplified sequence that were initially present in the sample. With the highly efficient detection chemistries, sensitive instrumentation, and optimized assays that are available today the number of DNA molecules of a particular sequence in a complex sample can be determined with unprecedented accuracy and sensitivity sufficient to detect a single molecule. Typical uses of real-time PCR include pathogen detection, gene expression analysis, single nucleotide polymorphism (SNP) analysis, analysis of chromosome aberrations, and most recently also protein detection by real-time immuno PCR.

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Available from: Amin Forootan, Oct 04, 2015
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    • "The result of primer pair No. 6 is not shown, since the formation of primer dimer was intensive and thus impeded a reliable quantification of target DNA by qPCR with SYBR green I. For primer pair No. 5, the level of detected DNA in all samples was on average one log 10 level lower than the level detected with primer pairs No. 3 and No. 4. This may be due to the length of the amplicon of primer pair No. 5 (770 bp) which may limit the efficiency of amplification and quantification by qPCR (Kubista et al., 2006), at least in the presence of PCR-inhibitors. This illustrates that the relative abundance of bacterial DNA might be biased by the use of primers with insufficient specificity and/or sensitivity for the DNA-target. "
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    ABSTRACT: Universal primers targeting the bacterial 16S-rRNA-gene allow quantification of the total bacterial load in variable sample types by qPCR. However, many universal primer pairs also amplify DNA of plants or even of archaea and other eukaryotic cells. By using these primers, the total bacterial load might be misevaluated, whenever samples contain high amounts of non-target DNA. Thus, this study aimed to provide primer pairs which are suitable for quantification and identification of bacterial DNA in samples such as feed, spices and sample material from digesters. For 42 primers, mismatches to the sequence of chloroplasts and mitochondria of plants were evaluated. Six primer pairs were further analysed with regard to the question whether they anneal to DNA of archaea, animal tissue and fungi. Subsequently they were tested with sample matrix such as plants, feed, feces, soil and environmental samples. To this purpose, the target DNA in the samples was quantified by qPCR. The PCR products of plant and feed samples were further processed for the Single Strand Conformation Polymorphism method followed by sequence analysis. The sequencing results revealed that primer pair 335F/769R amplified only bacterial DNA in samples such as plants and animal feed, in which the DNA of plants prevailed. Copyright © 2015. Published by Elsevier B.V.
    Journal of microbiological methods 04/2015; 113. DOI:10.1016/j.mimet.2015.04.001 · 2.03 Impact Factor
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    • "4.2. Multivariate gene expression profiling of L. monocytogenes in fermented sausages Although parametric tests, such as one-and two-way ANOVA, can provide an indication of what differences are significant between two or more conditions, when multiple samples, each containing the expression of multiple genes, are analyzed, the proper way is to use multivariate methods (Kubista et al., 2006; Bergkvist et al., 2008) since gene expressions tend to be correlated (violation of the parametric test assumption of independence). Multivariate gene expression profiling through PCA was run to classify the samples and genes, and to investigate the relationships between the variables. "
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    ABSTRACT: The current study reports a) the in situ transcriptional profiles of Listeria monocytogenes in response to fermented sausage stress and b) an approach in which in situ RT-qPCR data have been combined with advanced statistical techniques to discover potential stress resistance or cell viability biomarkers. Gene expression profiling of the pathogen has been investigated using RT-qPCR to understand how L. monocytogenes responds to the conditions encountered during the fermentation and ripening of sausages. A cocktail of five L. monocytogenes strains was inoculated into the batter of Cacciatore and Felino sausages. The RT-qPCR data showed that the acidic and osmotic stress-related genes were up-regulated. The transcripts of the lmo0669 gene increased during the fermentation and ripening of Cacciatore, whereas gbuA and lmo1421 were up-regulated during the ripening of Felino and Cacciatore, respectively. sigB expression was induced in both sausages throughout the whole process. Finally, the virulence-related gene prfA was down-regulated during the fermentation of Cacciatore. The multivariate gene expression profiling analysis suggested that sigB and lmo1421 or sigB and gbuA could be used as different types of stress resistance biomarkers to track, for example, stress resistance or cell viability in fermented sausages with short (Cacciatore) or long (Felino) maturation times, respectively. Copyright © 2014 Elsevier Ltd. All rights reserved.
    Food Microbiology 04/2015; 46:408-17. DOI:10.1016/ · 3.33 Impact Factor
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    • "At transcriptional level, several methods provide high sensitivity and accuracy in the quantification of gene expression, such as transcriptome sequencing, cDNA microarray, Northern blotting and quantitative real-time PCR (qPCR) technology (Kubista et al., 2006; Josefsen and Nielsen, 2011; Lang et al., 2014; Le et al., 2014). Owing to technical ease, low reagent cost, less hand-on time and high throughput , qPCR is increasingly and widely used to measure the expression of target genes across different samples (Kubista et al., 2006). "
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    ABSTRACT: In rubber tree, latex regeneration is one of the decisive factors influence the rubber yield, although its molecular regulation is not well known. Quantitative real-time PCR (qPCR) is a popular and powerful tool used to understand the molecular mechanisms of latex regeneration. However, the suitable reference genes required for qPCR are not available to investigate the expressions of target genes during latex regeneration. In this study, 20 candidate reference genes were selected and evaluated for their expression stability across the samples during the process of latex regeneration. All reference genes showed a relatively wide range of the threshold cycle values, and their stability was validated by four different algorithms (comparative delta Ct method, Bestkeeper, NormFinder and GeNorm). Three softwares (comparative delta Ct method, NormFinder and GeNorm) exported similar results that identify UBC4, ADF, UBC2a, eIF2 and ADF4 as the top five suitable references, and 18S as the least suitable one. The application of the screened references would improve accuracy and reliability of gene expression analysis in latex regeneration experiments. Copyright © 2015. Published by Elsevier B.V.
    Gene 03/2015; 563(2). DOI:10.1016/j.gene.2015.03.026 · 2.14 Impact Factor
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