Real-time PCR quantification using a variable reaction efficiency model

The quantitative real-time polymerase chain reaction (PCR) remains a cornerstone technique in gene expression analysis and sequence characterization. Despite the importance of the approach to experimental biology, the confident assignment of reaction efficiency to the early cycles of real-time PCR reactions remains problematic. Considerable noise may be generated when few cycles in the amplification are available to estimate peak efficiency. An alternate approach that uses data from beyond the log–linear amplification phase is explored in this article with the aim of reducing noise and adding confidence to efficiency estimates. PCR reaction efficiency is regressed to estimate the per-cycle profile of an asymptotically departed peak efficiency even when this is not closely approximated in the measurable cycles. The process can be repeated over replicates to develop a robust estimate of peak reaction efficiency. This leads to an estimate of the maximum reaction efficiency that may be considered primer design specific. Using a series of biological scenarios, we demonstrate that this approach can provide an accurate estimate of initial template concentration.     

Statistical analysis of real-time PCR data

Background  

Even though real-time PCR has been broadly applied in biomedical sciences, data processing procedures for the analysis of quantitative real-time PCR are still lacking; specifically in the realm of appropriate statistical treatment. Confidence interval and statistical significance considerations are not explicit in many of the current data analysis approaches. Based on the standard curve method and other useful data analysis methods, we present and compare four statistical approaches and models for the analysis of real-time PCR data.     

QPCR: Application for real-time PCR data management and analysis

Background  

Since its introduction quantitative real-time polymerase chain reaction (qPCR) has become the standard method for quantification of gene expression. Its high sensitivity, large dynamic range, and accuracy led to the development of numerous applications with an increasing number of samples to be analyzed. Data analysis consists of a number of steps, which have to be carried out in several different applications. Currently, no single tool is available which incorporates storage, management, and multiple methods covering the complete analysis pipeline.     

Accurate and efficient data processing for quantitative real-time PCR using a tripartite plant virus as a model

Real-time PCR is becoming a preferred method for quantification of minute amounts of nucleic acids. To achieve the full potential of this technique, accurate and convenient models for post-PCR data analysis are required. In this study, three different models were chosen to quantify the definitive copy numbers of Cucumber mosaic virus (CMV) genomic RNAs using raw fluorescence data of real-time PCR, and equations were proposed to compare their expression levels in virions or in planta. The results, as confirmed by standard curve and Northern blotting methods, show that the expression levels of different genes can be compared more accurately and more efficiently by these equations, especially using theoretical fluorescence (F0) and calibration factors (CF), determined by linear regression PCR (LinRegPCR). Thus, these equations, combined with data analysis by the LinRegPCR method, can greatly enhance the high-throughput quantification ability of real-time PCR, and permit accurate, reliable, and facile investigation of the changes in CMV RNAs accumulation.     

Correlation of serpin-protease expression by comparative analysis of real-time PCR profiling data

Imbalanced protease activity has long been recognized in the progression of disease states such as cancer and inflammation. Serpins, the largest family of endogenous protease inhibitors, target a wide variety of serine and cysteine proteases and play a role in a number of physiological and pathological states. The expression profiles of 20 serpins and 105 serine and cysteine proteases were determined across a panel of normal and diseased human tissues. In general, expression of serpins was highly restricted in both normal and diseased tissues, suggesting defined physiological roles for these protease inhibitors. A high correlation in expression for a particular serpin-protease pair in healthy tissues was often predictive of a biological interaction. The most striking finding was the dramatic change observed in the regulation of expression between proteases and their cognate inhibitors in diseased tissues. The loss of regulated serpin-protease matched expression may underlie the imbalanced protease activity observed in pathological states.     

Evaluation of real-time PCR data

If real-time PCR is to be of much worth to its user, some idea regarding the reliability of its data is essential. We discuss here some of the problems associated with interpreting numerical real-time PCR data that lend themselves to analytical evaluation. We translate into the language of molecular biology some of the criteria which are used to evaluate the performance of any new method (linearity, precision, specificity, limit of detection and quantification).     

A proportional analysis method using non-kinetic real-time PCR

Prompted by increasing interest in proportional analysis of genetic types, we developed a simple assay technique for determining the ratio of a specific target gene in the total genes that can be amplified with the same PCR primer. The key feature of this method is that the following two tasks are performed in a single-tube real-time PCR system: task 1, PCR amplification of the total genes including the target using a labeled PCR primer, with concurrent monitoring of the total copy number of the PCR product; task 2, detection of the signal of the target gene at each cycle of amplification, using a labeled nucleotide probe. In principle, the ratio of the target gene to the total genes is represented by the signal detected in 'task 2' at the cycle in which the PCR product reached a prescribed copy number (assessed by 'task 1').     

Standardized determination of real-time PCR efficiency from a single reaction set-up

We propose a computing method for the estimation of real-time PCR amplification efficiency. It is based on a statistic delimitation of the beginning of exponentially behaving observations in real-time PCR kinetics. PCR ground fluorescence phase, non-exponential and plateau phase were excluded from the calculation process by separate mathematical algorithms. We validated the method on experimental data on multiple targets obtained on the LightCycler platform. The developed method yields results of higher accuracy than the currently used method of serial dilutions for amplification efficiency estimation. The single reaction set-up estimation is sensitive to differences in starting concentrations of the target sequence in samples. Furthermore, it resists the subjective influence of researchers, and the estimation can therefore be fully instrumentalized.     

Molecular analysis of tetracycline-resistant gonococci: rapid detection of resistant genotypes using a real-time PCR assay

The aim of this study was to examine tetracycline-resistant gonococci and to set up a real-time PCR method to identify, in the same assay, both the chromosomally and the plasmid-mediated tetracycline-resistant genotypes. A retrospective analysis for tetracycline susceptibility was performed by the E -test and agar dilution methods on 289 gonococci isolated in Italy from 2003 to 2005. Molecular mechanisms of resistance were investigated by both sequence analyses of the three main genes associated with chromosomally mediated resistance ( mtrR , penB and rpsJ genes) and by the identification of plasmids carrying the tet M determinant associated with plasmid-mediated resistance, by PCR (American- or Dutch-type plasmids). The genetic relatedness of nonsusceptible strains was evaluated by pulsed field gel electrophoresis (PFGE). The results showed the presence of 22.5% tetracycline-resistant and 49.5% tetracycline-intermediate gonococci. Coexistence of chromosomally and plasmid-mediated resistance to tetracycline was observed in the majority of resistant isolates. No clonal structure was highlighted by analysis of PFGE pattern profiles. Real-time PCR assay was able to identify all the tetracycline nonsusceptible gonococci correctly for the presence of both chromosomally and/or plasmid-mediated genotypes.     

Mathematical model of real-time PCR kinetics

Several real-time PCR (rtPCR) quantification techniques are currently used to determine the expression levels of individual genes from rtPCR data in the form of fluorescence intensities. In most of these quantification techniques, it is assumed that the efficiency of rtPCR is constant. Our analysis of rtPCR data shows, however, that even during the exponential phase of rtPCR, the efficiency of the reaction is not constant, but is instead a function of cycle number. In order to understand better the mechanisms belying this behavior, we have developed a mathematical model of the annealing and extension phases of the PCR process. Using the model, we can simulate the PCR process over a series of reaction cycles. The model thus allows us to predict the efficiency of rtPCR at any cycle number, given a set of initial conditions and parameter values, which can mostly be estimated from biophysical data. The model predicts a precipitous decrease in cycle efficiency when the product concentration reaches a sufficient level for template-template re-annealing to compete with primer-template annealing; this behavior is consistent with available experimental data. The quantitative understanding of rtPCR provided by this model can allow us to develop more accurate methods to quantify gene expression levels from rtPCR data.     

Statistical methods for efficiency adjusted real-time PCR quantification

The statistical treatment for hypothesis testing using real-time PCR data is a challenge for quantification of gene expression. One has to consider two key factors in precise statistical analysis of real-time PCR data: a well-defined statistical model and the integration of amplification efficiency (AE) into the model. Previous publications in real-time PCR data analysis often fall short in integrating the AE into the model. Novel, user-friendly, and universal AE-integrated statistical methods were developed for real-time PCR data analysis with four goals. First, we addressed the definition of AE, introduced the concept of efficiency-adjusted Delta Delta Ct, and developed a general mathematical method for its calculation. Second, we developed several linear combination approaches for the estimation of efficiency adjusted Delta Delta Ct and statistical significance for hypothesis testing based on different mathematical formulae and experimental designs. Statistical methods were also adopted to estimate the AE and its equivalence among the samples. A weighted Delta Delta Ct method was introduced to analyze the data with multiple internal controls. Third, we implemented the linear models with SAS programs and analyzed a set of data for each model. In order to allow other researchers to use and compare different approaches, SAS programs are included in the Supporting Information. Fourth, the results from analysis of different statistical models were compared and discussed. Our results underline the differences between the efficiency adjusted Delta Delta Ct methods and previously published methods, thereby better identifying and controlling the source of errors introduced by real-time PCR data analysis.     

Chimerism analysis in sex-mismatched murine transplantation using quantitative real-time PCR

Marine experimental stem cell transplantations require the accurate discrimination and quantification of donor cells from host cells. A Y-chromosome-specific, quantitative real-time PCR (kinetic PCR) protocol for blood-derived DNA was developed. The assay sensitivity was extremely high with accurate detection of only 10 pg (six copies of Y target DNA) in a variable background of female DNA background ranging from 2.5 to 50 ng. The dynamic range of the assay provided accurate results ranging from 2.2 x 10(-2)% to 100% of male DNA in female background. The kinetic PCR assay can be used in all mouse strains, and a sample size as low as 2.5 ng total DNA is sufficient for analysis. Therefore, kinetic PCR allows engraftment kinetic studies on repeated blood draws of individual animals with no need for sacrifice. Compared to conventional PCR, the assay is much simplified, as neither the accurate adjustment of sample DNA concentration nor a post-reaction analysis procedure is required. The procedure is simple, free of radioactivity, and permits a throughput of 500-600 reactions per day.     

Relative quantification of mRNA: comparison of methods currently used for real-time PCR data analysis

Background  

Fluorescent data obtained from real-time PCR must be processed by some method of data analysis to obtain the relative quantity of target mRNA. The method chosen for data analysis can strongly influence results of the quantification.     

Murine maternal cell microchimerism: analysis using real-time PCR and in vivo imaging

In humans, maternal cells are present in the affected tissues of children with inflammatory myopathy, scleroderma, and neonatal lupus. It is unknown if maternal cell microchimerism (MCM) contributes to the pathology of disease. We sought to understand the factors that affect MCM to serve as a baseline for future mechanistic studies. Using a mouse model, we bred female mice transgenic for the luciferase (Luc) reporter gene to wild-type (WT) males. The WT offspring were sacrificed at various postnatal ages. DNA was extracted from multiple organs, and real-time PCR amplification was used to quantify Luc transgene as a marker for maternally derived cells. Sensitivity was one to two transgenic cells per 100,000 WT cells. MCM was noted in 85% of mice and 45% of tissues assayed. The average quantity of MCM was 158 maternal cells per 100,000 neonatal cells. The organs displaying the highest frequency and quantity of MCM were heart and lung (P < 0.001). Postnatal age up to 21 days did not appear to affect levels of MCM (P = 0.47), whereas increasing parity may increase levels of MCM. The data show that MCM is a common occurrence in healthy newborn mice, that it is present in their major organs, and that there are organ specific differences. This may represent differential migration of maternal cells or varying receptivity of specific fetal organs to microchimerism. Pregnancy history appears to play a role in maternal cell trafficking. The role of MCM in pregnancy and disease pathogenesis remains to be elucidated.