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Critical evaluation of methods used to determine amplification efficiency refutes the exponential character of real-time PCR.

Rutledge RG, Stewart D - BMC Mol. Biol. (2008)

Bottom Line: The most prominent approach is based on analysis of the "log-linear region", founded upon the presumption that amplification efficiency is constant within this region.This discrepancy was found to stem from misinterpreting the origin of the log-linear region, which is derived not from an invariant amplification efficiency, but rather from an exponential loss in amplification rate.In contrast, LRE analysis generated Emax estimates that correlated closely to that derived from a standard curve, despite the fact that standard curve analysis is founded upon exponential mathematics.

View Article: PubMed Central - HTML - PubMed

Affiliation: Natural Resources Canada, Canadian Forest Service, 1055 du PEPS, Quebec, Quebec G1V 4C7, Canada. Bob.Rutledge@NRCan.gc.ca

ABSTRACT

Background: The challenge of determining amplification efficiency has long been a predominant aspect of implementing real-time qPCR, playing a critical role in the accuracy and reliability that can be achieved. Based upon analysis of amplification profile position, standard curves are currently the gold standard for amplification efficiency determination. However, in addition to being highly resource intensive, the efficacy of this approach is limited by the necessary assumption that all samples are amplified with the same efficiency as predicted by a standard curve. These limitations have driven efforts to develop methods for determining amplification efficiency by analyzing the fluorescence readings from individual amplification reactions. The most prominent approach is based on analysis of the "log-linear region", founded upon the presumption that amplification efficiency is constant within this region. Nevertheless, a recently developed sigmoidal model has provided new insights that challenge such historically held views, dictating that amplification efficiency is not only dynamic, but is linearly coupled to amplicon DNA quantity. Called "linear regression of efficiency" or LRE, this kinetic-based approach redefines amplification efficiency as the maximal efficiency (Emax) generated at the onset of thermocycling.

Results: This study presents a critical evaluation of amplification efficiency determination, which reveals that potentially large underestimations occur when exponential mathematics is applied to the log-linear region. This discrepancy was found to stem from misinterpreting the origin of the log-linear region, which is derived not from an invariant amplification efficiency, but rather from an exponential loss in amplification rate. In contrast, LRE analysis generated Emax estimates that correlated closely to that derived from a standard curve, despite the fact that standard curve analysis is founded upon exponential mathematics. This paradoxical result implies that the quantitative efficacy of positional-based analysis relies not upon the exponential character of real-time PCR, but instead on the ability to precisely define the relative position of an amplification profile.

Conclusion: In addition to presenting insights into the sigmoidal character of the polymerase chain reaction, LRE analysis provides a viable alternative to standard curves for amplification efficiency determination, based on analysis of high-quality fluorescence readings within the central region of SYBR Green I generated amplification profiles.

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Related in: MedlinePlus

Amplification efficiency determination based on the log-linear region using a six cycle window. Log of the fluorescence readings within the lower region of the five amplification profiles presented in Figure 1A are plotted again cycle number, revealing a large log-linear region within all five profiles. The slope of this line provides an exponential-based estimate of amplification efficiency (Eloglin, equation 5) as summarized in the numerical inlay. The percentages listed below each data point show the predicted cycle efficiency derived from LRE analysis (equation 3). This predicts that a large loss in amplification efficiency occurs within the log-linear region.
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Figure 6: Amplification efficiency determination based on the log-linear region using a six cycle window. Log of the fluorescence readings within the lower region of the five amplification profiles presented in Figure 1A are plotted again cycle number, revealing a large log-linear region within all five profiles. The slope of this line provides an exponential-based estimate of amplification efficiency (Eloglin, equation 5) as summarized in the numerical inlay. The percentages listed below each data point show the predicted cycle efficiency derived from LRE analysis (equation 3). This predicts that a large loss in amplification efficiency occurs within the log-linear region.

Mentions: where Eloglin is the log-linear estimate of amplification efficiency. It is important to note, however, that the validity of this approach rests on the supposition that amplification efficiency remains constant throughout the log-linear region. Indeed, the very existence of a log-linear region lends itself to the compelling, albeit implicit, contention that amplification efficiency is invariant within this region. Nevertheless, critical examination reveals that linearity alone is insufficient to validate this contention. Figure 6 provides an example of this approach, based upon analysis of the five amplification profiles presented in Figure 1.


Critical evaluation of methods used to determine amplification efficiency refutes the exponential character of real-time PCR.

Rutledge RG, Stewart D - BMC Mol. Biol. (2008)

Amplification efficiency determination based on the log-linear region using a six cycle window. Log of the fluorescence readings within the lower region of the five amplification profiles presented in Figure 1A are plotted again cycle number, revealing a large log-linear region within all five profiles. The slope of this line provides an exponential-based estimate of amplification efficiency (Eloglin, equation 5) as summarized in the numerical inlay. The percentages listed below each data point show the predicted cycle efficiency derived from LRE analysis (equation 3). This predicts that a large loss in amplification efficiency occurs within the log-linear region.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2587475&req=5

Figure 6: Amplification efficiency determination based on the log-linear region using a six cycle window. Log of the fluorescence readings within the lower region of the five amplification profiles presented in Figure 1A are plotted again cycle number, revealing a large log-linear region within all five profiles. The slope of this line provides an exponential-based estimate of amplification efficiency (Eloglin, equation 5) as summarized in the numerical inlay. The percentages listed below each data point show the predicted cycle efficiency derived from LRE analysis (equation 3). This predicts that a large loss in amplification efficiency occurs within the log-linear region.
Mentions: where Eloglin is the log-linear estimate of amplification efficiency. It is important to note, however, that the validity of this approach rests on the supposition that amplification efficiency remains constant throughout the log-linear region. Indeed, the very existence of a log-linear region lends itself to the compelling, albeit implicit, contention that amplification efficiency is invariant within this region. Nevertheless, critical examination reveals that linearity alone is insufficient to validate this contention. Figure 6 provides an example of this approach, based upon analysis of the five amplification profiles presented in Figure 1.

Bottom Line: The most prominent approach is based on analysis of the "log-linear region", founded upon the presumption that amplification efficiency is constant within this region.This discrepancy was found to stem from misinterpreting the origin of the log-linear region, which is derived not from an invariant amplification efficiency, but rather from an exponential loss in amplification rate.In contrast, LRE analysis generated Emax estimates that correlated closely to that derived from a standard curve, despite the fact that standard curve analysis is founded upon exponential mathematics.

View Article: PubMed Central - HTML - PubMed

Affiliation: Natural Resources Canada, Canadian Forest Service, 1055 du PEPS, Quebec, Quebec G1V 4C7, Canada. Bob.Rutledge@NRCan.gc.ca

ABSTRACT

Background: The challenge of determining amplification efficiency has long been a predominant aspect of implementing real-time qPCR, playing a critical role in the accuracy and reliability that can be achieved. Based upon analysis of amplification profile position, standard curves are currently the gold standard for amplification efficiency determination. However, in addition to being highly resource intensive, the efficacy of this approach is limited by the necessary assumption that all samples are amplified with the same efficiency as predicted by a standard curve. These limitations have driven efforts to develop methods for determining amplification efficiency by analyzing the fluorescence readings from individual amplification reactions. The most prominent approach is based on analysis of the "log-linear region", founded upon the presumption that amplification efficiency is constant within this region. Nevertheless, a recently developed sigmoidal model has provided new insights that challenge such historically held views, dictating that amplification efficiency is not only dynamic, but is linearly coupled to amplicon DNA quantity. Called "linear regression of efficiency" or LRE, this kinetic-based approach redefines amplification efficiency as the maximal efficiency (Emax) generated at the onset of thermocycling.

Results: This study presents a critical evaluation of amplification efficiency determination, which reveals that potentially large underestimations occur when exponential mathematics is applied to the log-linear region. This discrepancy was found to stem from misinterpreting the origin of the log-linear region, which is derived not from an invariant amplification efficiency, but rather from an exponential loss in amplification rate. In contrast, LRE analysis generated Emax estimates that correlated closely to that derived from a standard curve, despite the fact that standard curve analysis is founded upon exponential mathematics. This paradoxical result implies that the quantitative efficacy of positional-based analysis relies not upon the exponential character of real-time PCR, but instead on the ability to precisely define the relative position of an amplification profile.

Conclusion: In addition to presenting insights into the sigmoidal character of the polymerase chain reaction, LRE analysis provides a viable alternative to standard curves for amplification efficiency determination, based on analysis of high-quality fluorescence readings within the central region of SYBR Green I generated amplification profiles.

Show MeSH
Related in: MedlinePlus