A new mathematical model for relative quantification in real-time RT-PCR.
TL;DR: This study enters into the particular topics of the relative quantification in real-time RT-PCR of a target gene transcript in comparison to a reference gene transcript and presents a new mathematical model that needs no calibration curve.
Abstract: Use of the real-time polymerase chain reaction (PCR) to amplify cDNA products reverse transcribed from mRNA is on the way to becoming a routine tool in molecular biology to study low abundance gene expression. Real-time PCR is easy to perform, provides the necessary accuracy and produces reliable as well as rapid quantification results. But accurate quantification of nucleic acids requires a reproducible methodology and an adequate mathematical model for data analysis. This study enters into the particular topics of the relative quantification in real-time RT–PCR of a target gene transcript in comparison to a reference gene transcript. Therefore, a new mathematical model is presented. The relative expression ratio is calculated only from the real-time PCR efficiencies and the crossing point deviation of an unknown sample versus a control. This model needs no calibration curve. Control levels were included in the model to standardise each reaction run with respect to RNA integrity, sample loading and inter-PCR variations. High accuracy and reproducibility (<2.5% variation) were reached in LightCycler PCR using the established mathematical model.
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TL;DR: This protocol provides an overview of the comparative CT method for quantitative gene expression studies and various examples to present quantitative gene Expression data using this method.
Abstract: Two different methods of presenting quantitative gene expression exist: absolute and relative quantification. Absolute quantification calculates the copy number of the gene usually by relating the PCR signal to a standard curve. Relative gene expression presents the data of the gene of interest relative to some calibrator or internal control gene. A widely used method to present relative gene expression is the comparative C(T) method also referred to as the 2 (-DeltaDeltaC(T)) method. This protocol provides an overview of the comparative C(T) method for quantitative gene expression studies. Also presented here are various examples to present quantitative gene expression data using this method.
20,580 citations
Cites methods from "A new mathematical model for relati..."
...The efficiency correction method calculates the relative expression ratio from the real-time PCR efficiencies and the C T (ref...
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TL;DR: The Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines target the reliability of results to help ensure the integrity of the scientific literature, promote consistency between laboratories, and increase experimental transparency.
Abstract: Background: Currently, a lack of consensus exists on how best to perform and interpret quantitative real-time PCR (qPCR) experiments. The problem is exacerbated by a lack of sufficient experimental detail in many publications, which impedes a reader’s ability to evaluate critically the quality of the results presented or to repeat the experiments.
Content: The Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines target the reliability of results to help ensure the integrity of the scientific literature, promote consistency between laboratories, and increase experimental transparency. MIQE is a set of guidelines that describe the minimum information necessary for evaluating qPCR experiments. Included is a checklist to accompany the initial submission of a manuscript to the publisher. By providing all relevant experimental conditions and assay characteristics, reviewers can assess the validity of the protocols used. Full disclosure of all reagents, sequences, and analysis methods is necessary to enable other investigators to reproduce results. MIQE details should be published either in abbreviated form or as an online supplement.
Summary: Following these guidelines will encourage better experimental practice, allowing more reliable and unequivocal interpretation of qPCR results.
12,469 citations
Cites background from "A new mathematical model for relati..."
...Clinical Chemistry 55:4 (2009) 617 parisontobeaccurate.Themostpopularmethodisnot necessarily the most appropriate, however, and alternative, more generalized quantitative models have been developed to correct for differences in amplification efficiency ( 62 ) and to allow the use of multiple reference genes (30)....
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TL;DR: Development and application of REST is explained, the usefulness of relative expression in real-time PCR using REST is discussed and the mathematical model used is based on the PCR efficiencies and the mean crossing point deviation between the sample and control group.
Abstract: Real-time reverse transcription followed by polymerase chain reaction (RT–PCR) is the most suitable method for the detection and quantification of mRNA. It offers high sensitivity, good reproducibility and a wide quantification range. Today, relative expression is increasingly used, where the expression of a target gene is standardised by a non-regulated reference gene. Several mathematical algorithms have been developed to compute an expression ratio, based on real-time PCR efficiency and the crossing point deviation of an unknown sample versus a control. But all published equations and available models for the calculation of relative expression ratio allow only for the determination of a single transcription difference between one control and one sample. Therefore a new software tool was established, named REST© (relative expression software tool), which compares two groups, with up to 16 data points in a sample and 16 in a control group, for reference and up to four target genes. The mathematical model used is based on the PCR efficiencies and the mean crossing point deviation between the sample and control group. Subsequently, the expression ratio results of the four investigated transcripts are tested for significance by a randomisation test. Herein, development and application of REST© is explained and the usefulness of relative expression in real-time PCR using REST© is discussed. The latest software version of REST© and examples for the correct use can be downloaded at http://www.wzw.tum.de/gene-quantification/.
7,196 citations
Cites background or methods from "A new mathematical model for relati..."
...It calculates the relative expression ratio on the basis of the PCR efficiency (E) and crossing point deviation (∆CP) of the investigated transcripts ( 6 ) and on a newly developed randomisation test macro....
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...Up to three CPs can be inserted in the table (run 1–3) per cDNA starting concentration and REST© determines the slope with a logarithmic algorithm, as published earlier (1, 6 ,14), as well as an indication of the linearity of this logarithmic alignment using Pearson’s correlation coefficient....
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...Equation 1 shows the most convenient mathematical model, which includes an efficiency correction for real-time PCR efficiency of the individual transcripts ( 6 )....
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...On the basis of the previously published mathematical model ( 6 ), REST© calculates the relative expression ratios on the basis of group means for target gene MT versus reference gene GAPDH and tests the group ratio results for significance....
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...The corresponding realtime PCR efficiency (E) of one cycle in the exponential phase was calculated according to the equation: E =1 0 [–1/slope] ,a s described earlier (1, 6 ,14)....
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TL;DR: The developed, and herein presented, software called BestKeeper determines the best suited standards, out of ten candidates, and combines them into an index, which can be compared with further target genes to decide, whether they are differentially expressed under an applied treatment.
Abstract: The stability of standard gene expression is an elementary prerequisite for internal standardisation of target gene expression data and many so called housekeeping genes with assumed stable expression can exhibit either upor down-regulation under some experimental conditions. The developed, and herein presented, software called BestKeeper determines the best suited standards, out of ten candidates, and combines them into an index. The index can be compared with further ten target genes to decide, whether they are differentially expressed under an applied treatment. All data processing is based on crossing points. The BestKeeper software tool was validated on four housekeeping genes and 10 members of the somatotropic axis differentially expressed in bovine corpora lutea total RNA. The BestKeeper application and necessary information about data processing and handling can be downloaded on http://www.wzw.tum.de/gene-quantification/bestkeeper.html
4,231 citations
Cites methods from "A new mathematical model for relati..."
...Reporting of the amount of target mRNA requires an accurate template preparation and relevant standardisation (Pfaffl 2001)....
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...This affects more advanced methods of gene expression study such as real-time PCR (Pfaffl 2001) or microarrays (Schuchhardt et al. 2000), as well as the traditional blotting methods....
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...This affects more advanced methods of gene expression study such as real-time PCR (Pfaffl 2001) or microarrays (Schuchhardt et al....
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...The slope of linear regression model fitted over log-transformed data of serially diluted input DNA concentrations plotted against their CPs (Rasmussen 2000, Pfaffl 2001)....
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TL;DR: Advanced and universally applicable models for relative quantification and inter-run calibration with proper error propagation along the entire calculation track are outlined in qBase, a free program for the management and automated analysis of qPCR data.
Abstract: Although quantitative PCR (qPCR) is becoming the method of choice for expression profiling of selected genes, accurate and straightforward processing of the raw measurements remains a major hurdle. Here we outline advanced and universally applicable models for relative quantification and inter-run calibration with proper error propagation along the entire calculation track. These models and algorithms are implemented in qBase, a free program for the management and automated analysis of qPCR data.
3,641 citations
Cites methods from "A new mathematical model for relati..."
...Depending on the settings, qBase will use the classic deltadelta-Ct method (100% PCR efficiency and one reference gene) [6], the Pfaffl modification of delta-delta-Ct (gene specific PCR efficiency correction and one reference gene) [7] or our generalized qBase model (gene specific PCR efficiency correction and multiple reference gene normalization)....
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...Pfaffl [7] modified the above model by adjusting for differences in PCR efficiency between the gene of interest (goi) and a reference gene (ref):...
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References
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Book•
15 Jan 2001
TL;DR: Molecular Cloning has served as the foundation of technical expertise in labs worldwide for 30 years as mentioned in this paper and has been so popular, or so influential, that no other manual has been more widely used and influential.
Abstract: Molecular Cloning has served as the foundation of technical expertise in labs worldwide for 30 years. No other manual has been so popular, or so influential. Molecular Cloning, Fourth Edition, by the celebrated founding author Joe Sambrook and new co-author, the distinguished HHMI investigator Michael Green, preserves the highly praised detail and clarity of previous editions and includes specific chapters and protocols commissioned for the book from expert practitioners at Yale, U Mass, Rockefeller University, Texas Tech, Cold Spring Harbor Laboratory, Washington University, and other leading institutions. The theoretical and historical underpinnings of techniques are prominent features of the presentation throughout, information that does much to help trouble-shoot experimental problems. For the fourth edition of this classic work, the content has been entirely recast to include nucleic-acid based methods selected as the most widely used and valuable in molecular and cellular biology laboratories. Core chapters from the third edition have been revised to feature current strategies and approaches to the preparation and cloning of nucleic acids, gene transfer, and expression analysis. They are augmented by 12 new chapters which show how DNA, RNA, and proteins should be prepared, evaluated, and manipulated, and how data generation and analysis can be handled. The new content includes methods for studying interactions between cellular components, such as microarrays, next-generation sequencing technologies, RNA interference, and epigenetic analysis using DNA methylation techniques and chromatin immunoprecipitation. To make sense of the wealth of data produced by these techniques, a bioinformatics chapter describes the use of analytical tools for comparing sequences of genes and proteins and identifying common expression patterns among sets of genes. Building on thirty years of trust, reliability, and authority, the fourth edition of Mol
215,169 citations
TL;DR: The technical aspects involved are discussed, conventional and kinetic RT-PCR methods for quantitating gene expression are contrasted, and the usefulness of these assays are illustrated by demonstrating the significantly different levels of transcription between individuals of the housekeeping gene family, glyceraldehyde-3-phosphate-dehydrogenase (GAPDH).
Abstract: The reverse transcription polymerase chain reaction (RT-PCR) is the most sensitive method for the detection of low-abundance mRNA, often obtained from limited tissue samples. However, it is a complex technique, there are substantial problems associated with its true sensitivity, reproducibility and specificity and, as a quantitative method, it suffers from the problems inherent in PCR. The recent introduction of fluorescence-based kinetic RT-PCR procedures significantly simplifies the process of producing reproducible quantification of mRNAs and promises to overcome these limitations. Nevertheless, their successful application depends on a clear understanding of the practical problems, and careful experimental design, application and validation remain essential for accurate quantitative measurements of transcription. This review discusses the technical aspects involved, contrasts conventional and kinetic RT-PCR methods for quantitating gene expression and compares the different kinetic RT-PCR systems. It illustrates the usefulness of these assays by demonstrating the significantly different levels of transcription between individuals of the housekeeping gene family, glyceraldehyde-3-phosphate-dehydrogenase (GAPDH).
4,100 citations
"A new mathematical model for relati..." refers background in this paper
...Nevertheless, the generation of stable and reliable standard material, either recombinant DNA or recombinant RNA, is very time consuming and it must be precisely quantified (2, 7 ,8)....
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...Therefore, quantification will always occur during the exponential phase, and it will not be affected by any reaction components becoming limited in the plateau phase ( 7 )....
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TL;DR: Results obtained with this approach indicate that a kinetic approach to PCR analysis can quantitate DNA sensitively, selectively and over a large dynamic range.
Abstract: We describe a simple, quantitative assay for any amplifiable DNA sequence that uses a video camera to monitor multiple polymerase chain reactions (PCRs) simultaneously over the course of thermocycling. The video camera detects the accumulation of double-stranded DNA (dsDNA) in each PCR using the increase in the fluorescence of ethidium bromide (EtBr) that results from its binding duplex DNA. The kinetics of fluorescence accumulation during thermocycling are directly related to the starting number of DNA copies. The fewer cycles necessary to produce a detectable fluorescence, the greater the number of target sequences. Results obtained with this approach indicate that a kinetic approach to PCR analysis can quantitate DNA sensitively, selectively and over a large dynamic range. This approach also provides a means of determining the effect of different reaction conditions on the efficacy of the amplification and so can provide insight into fundamental PCR processes.
2,366 citations
"A new mathematical model for relati..." refers methods in this paper
...The concept of threshold fluorescence is the basis of an accurate and reproducible quantification using fluorescence-based RT–PCR methods (22)....
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TL;DR: A novel approach to quantitative reverse transcriptase polymerase chain reaction (QC RT-PCR) using real time detection and the 5' nuclease assay has been developed and provides a convenient and high-throughput format for QC RT- PCR.
Abstract: A novel approach to quantitative reverse transcriptase polymerase chain reaction (QC RT-PCR) using real time detection and the 5' nuclease assay has been developed. Cystic fibrosis transmembrane transductance regulator (CFTR) target mRNA is reverse transcribed, amplified, detected, and quantitated in real time. A fluorogenic probe was designed to detect the CFTR amplicon. Relative increase in 6-carboxy-fluorescein reporter fluorescent emission is monitored during PCR amplification using an analytical thermal cycler. An internal control template containing the same primer sequences as the CFTR amplicon, but a different internal sequence, has been designed as a control. An internal control probe with a reporter fluorescent dye tetrachloro-6-carboxy-fluorescein was designed to hybridize to the internal control amplicon. The internal control template is placed in each reaction tube and is used for quantitative analysis of the CFTR mRNA. This method provides a convenient and high-throughput format for QC RT-PCR.
2,278 citations
"A new mathematical model for relati..." refers background in this paper
...A linear relationship between the CP, crossing the threshold fluorescence, and the log of the start molecules input in the reaction is given (18,23)....
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TL;DR: This quantitative PCR method provides a rapid and reliable way to quantify the amount of a specific mRNA in a sample of less than 0.1 ng of total RNA.
Abstract: A method for the quantitation of specific mRNA species by the polymerase chain reaction (PCR) has been developed by using a synthetic RNA as an internal standard. The specific target mRNA and the internal standard are coamplified in one reaction in which the same primers are used. The amount of mRNA is then quantitated by extrapolating against the standard curve generated with the internal standard. The synthetic internal standard RNA consists of a linear array of the sequences of upstream primers of multiple target genes followed by the complementary sequences to their downstream primers in the same order. This quantitative PCR method provides a rapid and reliable way to quantify the amount of a specific mRNA in a sample of less than 0.1 ng of total RNA. In addition, the same internal standard RNA is used, with appropriate primer pairs, to quantitate multiple different mRNA species.
1,609 citations