Efficiency of Real-Time PCR

Real-Time PCR is a technology that detects Polymerase Chain Reaction (PCR) amplification of a specific gene target automatically each cycle.  The real-time data enables the original gene target quantity in the PCR reaction to be deduced mathematically.

PCR can be divided into 3 phases (see figure 1).  The first phase may be termed geometric, exponential or logarithmic.  In this phase, PCR reagents are in excess, fueling consistent amplification efficiency.

The remarkable consistency of geometric amplification maintains the original quantitative relationships of the target gene across samples.  The geometric phase is also remarkable in that efficiency does not change with original gene quantity over a wide range.  For example, using highly concentrated target, such as plasmid, 20ml PCR reactions can produce a high quality standard curve spanning 9 logs or more.

During PCR, the DNA target may accumulate to a high enough level that one of the PCR reagents will no longer be sufficient to support geometric amplification.  At this point, geometric phase transitions to linear phase.  Linear phase is the second phase in PCR in which the efficiency declines cycle-to-cycle.  Changes in efficiency during linear phase become less and less consistent with increasing cycle number, so the data becomes less and less quantitative.

Eventually, PCR efficiency may become so low that there is no appreciable target amplification.  This final phase of PCR is called plateau.  Plateau phase data is not considered quantitative, unless special techniques are employed, such as those used for “digital PCR.”

Quantitative data can be acquired from the geometric phase using a variety of methods, such as baseline-threshold or relative-threshold.  Consequently, these geometric data points may have slightly different abbreviations, such as Ct, Crt or Cq, but they are all treated the same way in subsequent calculations.  For simplicity, the term “Ct” will be used in this document.

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PCR efficiency can be defined as the ratio of the number of target gene molecules at the end of a PCR cycle divided by the number of target molecules at the start of the same PCR cycle.  In the geometric phase, the efficiency is constant cycle-to-cycle.

Efficiency can be represented as a ratio or a percentage.  At maximum, a target sequence can double each cycle, because DNA only has two strands.  That efficiency can be represented as 2 or 100%.

In equation 1, efficiency (e) is a base in an exponential function, which means a change in the value of e can have a significant impact on the resulting quantity.  For example, for a Ct of 20, the quantities resulting from 100% vs. 80% efficiency differ by 8.2-fold.  Therefore, assigning accurate efficiency values is important for producing accurate quantity results.

In the 1990s, Perkin Elmer Corporation scientists developed a universal system for real-time PCR that consistently produced a geometric efficiency of 100%.  The system integrates universal cycling conditions, chemistry and assay design.  The proof of the system was demonstrated by a study of the efficiencies of 750 randomly selected TaqMan Gene Expression Assays (Amplification Efficiency of TaqMan Gene Expression Assays application note).

Real-time PCR is best performed when all assays have 100% geometric efficiency.  While it is possible to successfully run assays with less than 100% efficiency, lowered efficiency creates a list of issues that can add complexity and may cause problems.

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Thermo Fisher Scientific offers a number of options for real-time PCR assay design that conform to the universal system.  TaqMan Assays are off-the-shelf assays that have already been designed and tested.  Custom assays may be designed for gene targets not covered by TaqMan Assays.  Primer Express is desktop software that can be used to design custom assays and the Custom TaqMan Assay Design Tool is a web-based tool found on the Thermo Fisher Scientific website.

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To help maximize accuracy, the safest recommendation is to perform a geometric efficiency assessment on all involved assays.  Note that the geometric efficiency for TaqMan Assays is guaranteed to be 100% and it is extremely likely that Custom TaqMan Assays designed with either Primer Express or the Custom TaqMan Assay Design Tool will have 100% efficiency.  However, assays not designed in accordance with the universal system are at risk for lower efficiency.

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Using standard curves to assess efficiency entails additional cost and labor.

The ideal structure for a standard curve intended to assess efficiency is 7-points with a 10-fold series (see Amplification Efficiency of TaqMan Gene Expression Assays application note).  Such a dilution series would require highly concentrated target, which may not be available naturally in samples.

Errors in standard curve slopes are very common.  Many factors can cause such errors, including inhibitors, contamination, pipet precision error, pipet calibration error and dilution point mixing problems.  These errors explain how slopes that theoretically correspond to greater than 100% efficiency could occur, even though geometric efficiency cannot exceed 100%.  Therefore, any efficiency assessments based on equation 2 should be taken with a great deal of caution.

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One factor that has a predictable, mathematical impact on standard curve slopes is pipet calibration error.  A method to correct for potential pipet calibration error is to subtract the slopes of two standard curves that were generated from the same dilution series. This method is shown in a Perkin Elmer Corporation document called User Bulletin #2. Theoretically, if two assays have the same geometric efficiency, the difference in the two standard curve slopes should be zero.  If the efficiencies are equal, they are most likely 100%, because an assay that is not 100% efficient will have a random efficiency, making it highly unlikely the two assays will have equal efficiencies.

However, subtracting slopes does not correct for the other types of errors, so the User Bulletin #2 method is still error prone.

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The basis of real-time quantitative PCR is constant geometric efficiency, which means that in an amplification plot with a log y-axis scale, all the geometric slopes should be parallel within a given assay.  If multiple assays have 100% geometric efficiency, the efficiencies are equal, which means geometric slopes should be parallel inter-assay as well.  Such parallelism is in fact observed (see figure 2).

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The original quantification method for real-time PCR involved standard curves.  A standard curve was run for each assay on the plate, best fit line equations were calculated for each standard curve and Ct values were transformed into quantities based on those line equations.

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The ΔΔCt method is a method to quantify using real-time PCR data that does not require standard curves.  The ΔΔCt method has multiple variants.  In the traditional version, transformation of Ct values into quantities occurs as the final step.  Steps such as normalizing to a normalizer gene or a calibrator sample are done earlier by subtracting Ct values, producing ΔCt or ΔΔCt values (see equation 4).

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The answer is no.  PCR related inhibition falls into 3 categories:  reverse transcription inhibition, solubility inhibition and Taq DNA polymerase inhibition.  For those chemistries involving a traditional retrovirus-derived reverse transcriptase, inhibiting that enzyme increases any resulting Ct value, but has no impact on Taq DNA polymerase activity.  Solubility inhibition reduces the amount of DNA target available for amplification, resulting in a Ct increase, but the geometric phase is unaffected.  Taq inhibition reduces the concentration of active enzyme the reaction, which may cause the geometric phase to prematurely transition to linear phase.  In worst case scenario, Taq inhibition may make detection of the geometric phase very difficult, but geometric phase efficiency remains the same.

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