A new real-time PCR method to overcome significant quantitative inaccuracy due to slight amplification inhibition
- Michele Guescini†1, 2Email author,
- Davide Sisti†3,
- Marco BL Rocchi3,
- Laura Stocchi1 and
- Vilberto Stocchi1, 2Email author
© Guescini et al; licensee BioMed Central Ltd. 2008
Received: 21 April 2008
Accepted: 30 July 2008
Published: 30 July 2008
Real-time PCR analysis is a sensitive DNA quantification technique that has recently gained considerable attention in biotechnology, microbiology and molecular diagnostics. Although, the cycle-threshold (Ct) method is the present "gold standard", it is far from being a standard assay. Uniform reaction efficiency among samples is the most important assumption of this method. Nevertheless, some authors have reported that it may not be correct and a slight PCR efficiency decrease of about 4% could result in an error of up to 400% using the Ct method. This reaction efficiency decrease may be caused by inhibiting agents used during nucleic acid extraction or copurified from the biological sample.
We propose a new method (Cy 0 ) that does not require the assumption of equal reaction efficiency between unknowns and standard curve.
The Cy 0 method is based on the fit of Richards' equation to real-time PCR data by nonlinear regression in order to obtain the best fit estimators of reaction parameters. Subsequently, these parameters were used to calculate the Cy 0 value that minimizes the dependence of its value on PCR kinetic.
The Ct, second derivative (Cp), sigmoidal curve fitting method (SCF) and Cy 0 methods were compared using two criteria: precision and accuracy. Our results demonstrated that, in optimal amplification conditions, these four methods are equally precise and accurate. However, when PCR efficiency was slightly decreased, diluting amplification mix quantity or adding a biological inhibitor such as IgG, the SCF, Ct and Cp methods were markedly impaired while the Cy 0 method gave significantly more accurate and precise results.
Our results demonstrate that Cy 0 represents a significant improvement over the standard methods for obtaining a reliable and precise nucleic acid quantification even in sub-optimal amplification conditions overcoming the underestimation caused by the presence of some PCR inhibitors.
In the last few years, the real-time polymerase chain reaction (PCR) has rapidly become the most widely used technique in modern molecular biology [1–4]. This technique relies on fluorescence-based detection of amplicon DNA and allows the kinetics of PCR amplification to be monitored in real time, making it possible to quantify nucleic acids with extraordinary ease and precision. With a large dynamic range (7–8 magnitudes) and a high degree of sensitivity (5–10 molecules), the real-time PCR addresses the evident requirement for quantitative data analysis in molecular medicine, biotechnology, microbiology and diagnostics [5, 6].
Although, the real-time PCR analysis has gained considerable attention in many fields of molecular biology, it is far from being a standard assay. One of the problems associated with this assay, which has a direct impact on its reliability, is inconsistent data analysis. At the present, real-time PCR analysis is highly subjective and, if carried out inappropriately, confuses the actual results . Many different options for data processing are currently available. The basic choice in real time PCR calculations is between absolute quantification, based on standard curve, and relative quantification, based on PCR efficiency calculation. Using the software currently available, analysis of real-time PCR data is generally based on the "cycle-threshold" method. The cycle-threshold is defined as the fractional cycle number in the log-linear region of PCR amplification in which the reaction reaches fixed amounts of amplicon DNA. There are two methods for determining the cycle-threshold value; one method, namely fit point, is performed by drawing a line parallel to the x-axis of the real-time fluorescence intensity curve (Ct) . The second, namely second derivative, calculates the fractional cycle where the second derivative of the real-time fluorescence intensity curve reaches the maximum value (Cp) . Standard curve method requires generating serial dilutions of a given sample and performing multiple PCR reactions on each dilution [10, 11], the threshold-cycle values are then plotted versus the log of the dilution and a linear regression is performed from which the mean efficiency can be derived. This approach is only valid if the threshold-cycle values are measured from the exponential phase of the PCR reaction and if the efficiency is identical between amplifications. Furthermore, this efficiency is assumed to be the same for all the standard dilutions, but some authors have reported that this assumption may be questionable .
It is well-recognized that template quality is one of the most important determinants of real-time PCR reliability and reproducibility , and numerous authors have shown the significant reduction in the sensitivity and kinetics of real-time PCR assays caused by inhibitory components frequently found in biological samples [14–17]. The inhibiting agents may be reagents used during nucleic acid extraction or copurified components from the biological sample such as bile salts, urea, haeme, heparin, and immunoglobulin G. Inhibitors can generate strongly inaccurate quantitative results; while, a high degree of inhibition may even create false-negative results.
The Ct method is the most widely used method even though its calculation is user-dependent. The Ct method is quite stable and straightforward but the accuracy of estimates is strongly impaired if efficiency is not equal in all reactions. Indeed, uniform reaction efficiency is the most important assumption of the Ct method.
An alternative approach, proposed by Liu and Saint , assumes a dynamic change in efficiency fitting PCR amplification with a sigmoid function (S igmoidal c urve f itting method, SCF). One of the advantages of this regression analysis is that it allows us to estimate the initial template amount directly from the non-linear regression, eliminating the need for a standard curve. These pioneering works showed that it was possible to obtain absolute quantification from real-time fluorescence curve shape. However, recent reports have demonstrated that, in an optimized assay, the Ct method remains the gold standard due to the inherent errors of the multiple estimates used in non-linear regression [19, 20].
We propose, in this report, a modified standard curve-based method (named Cy 0 ) that does not require the assumption of uniform reaction efficiency between standards and unknown and does not involve any choice of threshold level by the user.
The aim of this work was also to compare the accuracy and precision of the SCF, Ct, Cp and Cy 0 methods in presence of varying PCR kinetics. Our results clearly show that the proposed data processing procedure can effectively be applied in the quantification of samples characterized by slight amplification inhibition obtaining reliable and precise results.
The absolute quantification method relies on the comparison of distinct samples, such as the comparison of a biological sample with a standard curve of known initial concentration . We wondered how accuracy and precision change when a standard curve is compared with unknown samples characterized by different efficiencies. A natural way of studying the effect of efficiency differences among samples on quantification would be to compare the amounts of a quantified gene.
A slight amplification inhibition in the quantitative real-time PCR experiments was obtained by using two systems: decreasing the amplification mix used in the reaction and adding varying amounts of IgG, a known PCR inhibitor.
For the first system, we amplified the MT-ND1 gene by real-time PCR in reactions having the same initial amount of DNA but different amounts of SYBR Green I Master mix. A standard curve was performed over a wide range of input DNA (3.14 × 107–3.14 × 101) in the presence of optimal amplification conditions (100% amplification mix), while the unknowns were run in the presence of the same starting DNA amounts but with amplification mix quantities ranging from 60% to 100%. This produced different reaction kinetics, mimicking the amplification inhibition that often occurs in biological samples [17, 22].
Furthermore, quantitative real-time PCR quantifications were performed in the presence of an optimal amplification reaction mix added with serial dilutions of IgG (0.0625 – 2 μg/ml) thus acting as the inhibitory agent .
The reaction efficiency obtained was estimated by the LinReg method . This approach identifies the exponential phase of the reaction by plotting the fluorescence on a log scale. A linear regression is then performed leading to the estimation of the efficiency of each PCR reaction.
Quantitative Real-Time PCR
The DNA standard consisted of a pGEM-T (Promega) plasmid containing a 104 bp fragment of the mitochondrial gene NADH dehydrogenase 1 (MT-ND1) as insert. This DNA fragment was produced by the ND1/ND2 primer pair (forward ND1: 5'-ACGCCATAAAACTCTTCACCAAAG-3' and reverse ND2: 5'-TAGTAGAAGAGCGATGGTGAGAGCTA-3'). This plasmid was purified using the Plasmid Midi Kit (Qiagen) according to the manufacturer's instructions. The final concentration of the standard plasmid was estimated spectophotometrically by averaging three replicate A260 absorbance determinations.
Real time PCR amplifications were conducted using LightCycler® 480 SYBR Green I Master (Roche) according to the manufacturer's instructions, with 500 nM primers and a variable amount of DNA standard in a 20 μl final reaction volume. Thermocycling was conducted using a LightCycler® 480 (Roche) initiated by a 10 min incubation at 95°C, followed by 40 cycles (95°C for 5 s; 60°C for 5 s; 72°C for 20 s) with a single fluorescent reading taken at the end of each cycle. Each reaction combination, namely starting DNA and amplification mix percentage, was conducted in triplicate and repeated in four separate amplification runs. All the runs were completed with a melt curve analysis to confirm the specificity of amplification and lack of primer dimers. Ct (fit point method) and Cp (second derivative method) values were determined by the LightCycler® 480 software version 1.2 and exported into an MS Excel data sheet (Microsoft) for analysis after background subtraction (available as Additional file 1). For Ct (fit point method) evaluation a fluorescence threshold manually set to 0.5 was used for all runs.
Description of the SCF method
where F 0 represents the initial target quantity expressed in fluorescence units. Conversion of F 0 to the number of target molecules was obtained by a calibration curve in which the log input DNA was related to the log of F 0 . Subsequently, this equation was used for quantification with log transformation of fluorescence data to increase goodness-of-fit as described in Goll et al. 2006 .
Description of the Cy 0 method
where x is the cycle number, F x is the reaction fluorescence at cycle x, F max is the maximal reaction fluorescence, x is the fractional cycle of the turning point of the curve, d represents the Richards coefficient, and F b is the background reaction fluorescence. The inflection point coordinate (Flex) was calculated as follows (Additional file 2):
Although the Cy 0 is a single quantitative entity, as is the Ct or Cp for threshold methodologies, it accounts for the reaction kinetic because it is calculated on the basis of the slope of the inflection point of fluorescence data.
Statistical data analysis
Nonlinear regressions (for 4-parameter sigmoid and 5-parameter Richards functions) were performed determining unweighted least squares estimates of parameters using the Levenberg-Marquardt method. Accuracy was calculated using the following equation:
, where was the relative error, while and were the estimated and the true number of DNA molecules for each combination of input DNA (n Dna ) and amplification mix percentage (% mix ) used in the PCR. Precision was calculated as:
, where was the coefficient of variation, and were the mean and the standard deviation for each combination of nDna and %mix. In order to verify that the Richards curves, obtained by nonlinear regression of fluorescence data, were not significantly different from the sigmoidal curves, the values of d parameter were compared to the expected value d = 1, using t test for one sample. For each combination of n Dna , % mix , the t values were calculated as follow:
, where and were the mean and the standard error of d values for each combination of n Dna and % mix , with p(t) < 0.05 for significance level. values were reported using 3-d scatterplot graphic, a complete second order polinomial regression function was shown to estimate the trend of accuracy values. where also reported using 3-d contour plots using third-order polynomials spline fitting. All elaborations and graphics were obtained using Excel (Microsoft), Statistica (Statsoft) and Sigmaplot 10 (Systat Software Inc.).
Experimental system 1: reduction of amplification mix percentage
Precision and accuracy of the SCF method
Previous studies have shown that the SCF approach can lead to quantification without prior knowledge of amplification efficiency [18, 19, 26]; therefore, we evaluated the performance of this method on our data set. To assess the effect of unequal efficiencies on accuracy, the calculated input DNA, expressed as molecular number, was compared to the expected value obtaining the relative error (RE). The precision was further evaluated measuring the variation coefficient (CV%) of the estimated initial DNA in the presence of different PCR efficiencies and input DNA.
In our experimental design, the SCF method showed a very poor precision (mean CV% = 594.74%) and low accuracy (mean RE = -5.05). The impact of amplification efficiency decline on accuracy was very strong resulting in an underestimate of samples of up to 500% (Additional file 3). The log transformation of fluorescence data before sigmoidal fitting significantly reduced the CV% and RE to 66.12% and -0.20, respectively; however, the overall bias remained the same . Finally, we also tested an improved SCF approach based on a previous study by Rutledge 2004  without obtaining significant amelioration (Additional file 4).
The Cy 0 method
Comparison of five S-shaped models to fit the PCR curve: Sigmoid, Richards, Gompertz, Hill and Chapman.
Adj R 2
Standard Error of Estimate
f = F b +F max /(1+exp(-(x-c)/b))
f = F b +(F max /(1+exp(-(1/b)*(x-c)))^d)
f = F b +F max *exp(-exp(-(x-c)/b))
f = F b +F max *x^b/(d^b+x^b)
f = F b +F max *(1-exp(-b*x))^d
t statistic values obtained for all variable combinations.
Amplification mix percentage
Log 10 input DNA
Precision and accuracy of the Ct, Cp and Cy 0 methods
Comparison of mean Relative Error and mean Variation Coefficient among the Ct, Cp, Cy 0 and SCF methods.
Log 10 SCF
Experimental system 2: Real-time PCR quantification in the presence of the inhibitor IgG
None of the current quantitative PCR data treatment methods is in fact fully assumption-free, and their statistical reliability are often poorly characterized. In this study, we evaluated whether known real-time elaboration methods could estimate the amount of DNA in biological samples with precision and accuracy when reaction efficiencies of the unknown are different from those of the standard curve.
Our experimental systems consisted in the quantification of samples with the same known starting template amount but the amplification reaction, performed for the real-time PCR assay, had a slightly decreasing efficiency. This is clearly not in agreement with the main assumption of the threshold approach which holds that the amplification efficiency of samples has to be identical to, or not significantly different from, that predicted by the standard curve. However, such an assumption has been reported to be patently invalid for many cases in medical diagnostics. In fact, some, if not all, of the biological samples may contain inhibitors that are not present in the standard nucleic acid samples used to construct the calibration curve, leading to an underestimation of the DNA quantities in the unknown samples [28, 29]. In our study, slightly decreasing efficiencies were obtained by two systems: diluting the master enzyme mix or adding IgG, a known inhibitor of PCR. Although, the first system is an "in vitro" simulation of PCR inhibition, it produces amplification curves very similar to those obtained in the presence of a biological inhibitor like IgG.
Notably, our experimental setup is not characterized by aberrant amplification reactions. On the contrary, the reactions show a slight mean efficiency decrease which is always the case of biological samples. This PCR inhibition remains undetected when using a threshold approach leading to target underestimation. Moreover, small differences in amplification efficiency produce large quantitative errors and the frequency and magnitude of these errors are virtually impossible to ascertain using a threshold approach. It has been shown that a difference as small as 4% in PCR efficiency could translate into a 400% error in comparative Ct method based quantification .
Considering previous works [18, 19] which demonstrated the capability of the SCF method to quantify a sample without prior knowledge of amplification efficiency, our first choice was to process the experimental data by the SCF method. The effectiveness of the SCF approach is based on curve fitting of raw data so that variations unique to each amplification reaction are incorporated into the analysis. Hence, the results reported herein surprisingly demonstrated that the accuracy and precision of the SCF method was markedly impaired when efficiency fell. In fact, when PCR efficiency decreased by about 2.5% (88.8% efficiency value in the presence of 100% of the amplification mix dropped to 84.4% efficiency in the presence of 60% of the mix), we observed, using the SCF method with log-transformation, that the RE and CV went from 15% to 43% and from 61% to 55%, respectively.
Furthermore, we found that, when the amplification curve was inhibited, by IgG, the method proposed by Rutledge  can not be applied because for each cut-off cycle eliminated from the plateau phase the F 0 value progressively decreased without ever reaching a minimum value. These observations are in agreement with two recent studies, which reported that it is possible to obtain absolute quantification from real-time data without a standard curve, but the Ct method remains a gold standard due to the inherent errors of the multiple estimates used in nonlinear regression [19, 20]. These observations are in accordance with Feller's conclusions that different S-shaped curves can be effectively fitted with various sigmoid models , each providing distinct F 0 values. Thus sigmoid fit methods such as the logistic model, used in the SCF approach, are purely descriptive and quantitative results may be unreliable. This led us to develop a new mathematical data treatment method, named Cy 0 , based on nonlinear regression fitting of real-time fluorescence data. The proposed method's main advantages are its use of the Richards equation for obtaining the coordinate of the inflection point and the determination of the quantitative entity Cy 0 using the five parameters of reaction curve.
Although the logistic growth equation generates a curve that tends towards an exponential form at low fluorescence values, making this curve ideal to model PCR reaction, its maximum slope, or inflection point, is always imposed to be at half the value of the upper asymptote, (F max -F b )/2. This is unsatisfactory because the factors that determine the growth rate are complex and some amplification systems, although characterized by good reaction efficiency, as assessed by standard curve, do not have the center of symmetry in the inflection point. The Richards equation is a more flexible growth function because it has an additional parameter, which is a shape parameter that can make the Richards equation equivalent to the logistic, Gompertz, or monomolecular equations [31, 32]. Variation of the shape parameter allows the point of inflection of the curve to be at any value between the minimum and the upper asymptote; when d = 1 the Eq. 3 becomes the sigmoidal equation.
Furthermore, since very small errors of the multiple estimates used in non-linear regression lead to large variations in F 0 values, the real-time PCR kinetic parameters were used to define a new quantitative entity, the Cy 0 . The Cy 0 relies on the inflection point position and on the slope of the fluorescence curve at that point, so that its value slightly changes in relation to PCR efficiency. In particular, in a slightly inhibited amplification reaction, the fluorescence curves are shifted towards the right and/or they are less steep; this generates higher Ct values than those found under optimal amplification conditions, underestimating the target amount. In the Cy 0 method, the tangents, calculated from different PCR efficiency, tend to intersect at a common point near the x-axis leading to small variations in the Cy 0 values (Fig. 4).
The standard curve approach was chosen for the proposed method because currently there no genuine mathematical model for PCR efficiency assessment. The main complication is that actual efficiency amplification is not constant through the PCR run being high in exponential phase and gradually declining towards the plateau phase [33–35]. However, most current methods of PCR efficiency assessment report "overall" efficiency as a single value [13, 24, 36, 37]. Moreover, recent publications on PCR efficiency assessment have concentrated on the analysis of individual shapes of fluorescence plots in order to estimate a dynamic efficiency value [19, 20, 27, 38]. This proliferation of new methods to assess PCR efficiency demonstrates that, at present, there is not an accepted procedure to evaluate PCR efficiency from a single run, hence some methods can "overestimate" and others "underestimate" the "true" PCR efficiency . In contrast, the standard curve method is based on a simple approximation of data obtained in standard dilutions to unknown samples. In this procedure PCR efficiency assessment is based on the slope of the standard curve. Indeed, the original method (Ct) does not account for PCR efficiencies in individual target samples. The proposed procedure overcomes this limitation by evaluating single amplification variations using Richards curve fitting and subsequently produces a Cy 0 value that minimizes the dependence of its value on PCR kinetic.
We then compared our method with the Ct method, the actual "gold standard" in real-time PCR quantification and the Cp method which is also used in molecular diagnostics. Both methods are based on standard curve methodology and are the most frequently used in this field. The Ct, Cp and Cy 0 methods were evaluated on the same data set using two criteria: precision and accuracy. We defined the accuracy of a model as its ability to provide expected concentrations of the known dilutions under different PCR amplification efficiencies. On the contrary, precision is related to the variability of the results obtained from a given model, and it indicates whether reliable results may be obtained from a small data collection. Our results clearly demonstrated that, under optimal amplification conditions, these three methods were equally precise and accurate. However, when the PCR efficiency decreased, due to amplification mix dilution or IgG presence, the Ct method was markedly impaired and the Cp and Cy 0 methods proved to be significantly more accurate than the Ct method. Notably, the Cy 0 method showed accuracy levels higher than the Cp method maintaining the same precision.
The ability to carry out reliable nuclei acid quantification even in sub-optimal amplification conditions is particularly useful when PCR optimization is not possible, as in the case of high-throughput screening of gene expression or biological samples difficult to cleanse of PCR inhibitors.
Furthermore, the Cy 0 method is completely objective and assumption-free. Indeed, it does not require the choice of a threshold value and the assumption of similar amplification efficiency between the standard curve and biological samples, necessary in the Ct method. Moreover, there is no need to assume that base pair composition and amplicon size do not impact the fluorescence characteristics of SYBR Green I, required in optical calibration methods like SCF . Our procedure may have future applications in TaqMan assays, where the Taq DNA polymerase digests a probe labelled with a fluorescent reporter and quencher dye and the signal diverges from the product resulting in non-symmetric amplification curves that can be effectively modelled by Richards equation . Further work is needed to extensively verify the accuracy and precision of the Cy 0 method in the presence of other known PCR inhibitors like phenol, haemoglobin, fat and tannic acid [17, 22].
Real-time PCR analysis is becoming increasingly important in biomedical research because of its accuracy, sensitivity and high efficiency. Although, real-time PCR analysis has gained considerable attention, it is far from being a standard assay. The standard methods are quite stable and straightforward but the accuracy of estimates is strongly impaired if efficiency is not equal in all reactions. Furthermore, the assumption of uniform efficiency has been reported to be invalid in many cases regarding medical diagnostics. In fact, the biological samples may contain inhibitors that could lead to different amplification efficiencies among samples.
We propose, in this report, a modified standard curve-based method, called Cy 0 , that does not require the assumption of uniform reaction efficiency between standards and unknown.
To the best of our knowledge, this is the first method in which the stability and reliability of a standard curve approach is combined with a fitting procedure to overcome the key problem of PCR efficiency determination in real-time PCR nucleic acid quantification. The data reported herein clearly show that the Cy 0 method is a valid alternative to the standard method for obtaining reliable and precise nucleic acid quantification even in sub-optimal amplification conditions, such as those found in the presence of biological inhibitors like IgG.
coefficient of variation
sigmoidal curve fitting.
We thank Dr. Pasquale Tibollo for technical assistance and Dr. Giosuè Annibalini for helpful comments on the manuscript.
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