Limits...
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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Assessing the impact of the fluorescence threshold on slope-derived determination of amplification efficiency. (A) Amplification profiles taken from Figure 1A with the red lines representing the fluorescence thresholds that were manually set to values that span the entire height of the amplification profiles. (B) Standard curves generated from each of the five Ft settings (increasing from left to right). The numerical inlay summarizes the linear regression analysis, which indicates that Ft has no major impact on either r2 or Eslope until placed into the extreme upper region of the amplification profiles.
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Figure 2: Assessing the impact of the fluorescence threshold on slope-derived determination of amplification efficiency. (A) Amplification profiles taken from Figure 1A with the red lines representing the fluorescence thresholds that were manually set to values that span the entire height of the amplification profiles. (B) Standard curves generated from each of the five Ft settings (increasing from left to right). The numerical inlay summarizes the linear regression analysis, which indicates that Ft has no major impact on either r2 or Eslope until placed into the extreme upper region of the amplification profiles.

Mentions: The general validity of this presumption can be tested empirically by compiling a series of standard curves generated by progressively increasing Ft. This also allows testing of another general presumption, which is that the integrity of a standard curve relies on placing Ft within this putative exponential region [14,15,28,29]. As illustrated in Figure 2, this appears not to be the case, in that Eslope was unaffected until the fluorescence threshold was placed into the extreme upper region of the profiles.


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)

Assessing the impact of the fluorescence threshold on slope-derived determination of amplification efficiency. (A) Amplification profiles taken from Figure 1A with the red lines representing the fluorescence thresholds that were manually set to values that span the entire height of the amplification profiles. (B) Standard curves generated from each of the five Ft settings (increasing from left to right). The numerical inlay summarizes the linear regression analysis, which indicates that Ft has no major impact on either r2 or Eslope until placed into the extreme upper region of the amplification profiles.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Assessing the impact of the fluorescence threshold on slope-derived determination of amplification efficiency. (A) Amplification profiles taken from Figure 1A with the red lines representing the fluorescence thresholds that were manually set to values that span the entire height of the amplification profiles. (B) Standard curves generated from each of the five Ft settings (increasing from left to right). The numerical inlay summarizes the linear regression analysis, which indicates that Ft has no major impact on either r2 or Eslope until placed into the extreme upper region of the amplification profiles.
Mentions: The general validity of this presumption can be tested empirically by compiling a series of standard curves generated by progressively increasing Ft. This also allows testing of another general presumption, which is that the integrity of a standard curve relies on placing Ft within this putative exponential region [14,15,28,29]. As illustrated in Figure 2, this appears not to be the case, in that Eslope was unaffected until the fluorescence threshold was placed into the extreme upper region of the profiles.

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