Result Variation and Efficiency Kinetics in Real-Time PCR

  • Reza Shahsiah Department of Pathology, Imam Hospitals Complex, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran.
  • Alireza Abdollahi Department of Pathology, Imam Hospitals Complex, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran.
  • Farid Azmoudeh Ardalan Department of Pathology, Imam Hospitals Complex, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran.
  • Mohammad Taghi Haghi-Ashtiani Pediatrics Center of Excellence, Children's Medical Center, Tehran University of Medical Sciences, Tehran, Iran.
  • Issa Jahanzad Department of Pathology, Imam Hospitals Complex, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran.
  • Mohsen Nassiri Toosi Department of Gastroenterology, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran.
Keywords: Polymerase chain reaction, efficiency, regression analysis, logistic model

Abstract

Fluorescent monitoring of DNA amplification is the basis of real-time PCR. Absolute quantification can be achieved using a standard curve method. The standard curve is constructed by amplifying known amounts of standards under identical conditions to that of the samples.The objective of the current study is to propose a mathematical model to assess the acceptability of PCR resulys.Four commercial standards for HCV-RNA (hepatitis C virus RNA) along with 6 patient samples were measured by real-time PCR, using two different RT-PCR reagents. The standard deviation of regression (Sy,x) was calculated for each group of standard and compared by F-Test. The efficiency kinetics was computed by logistic regression, c2 goodness of fit test was preformed to assess the appropriateness of the efficiency curves.Calculated efficiencies were not significantly different from the value predicted by logistic regression model. Reactions with more variation showed less stable efficiency curves, with wider range of amplification efficiencies.Amplification efficiency kinetics can be computed by fitting a logistic regression curve to the gathered fluorescent data of each reaction. This model can be employed to assess the acceptability of PCR results calculated by standard curve method.

References

Kubista M, Andrade JM, Bengtsson M, Forootan A, Jonák J, Lind K, et al. The real-time polymerase chain reaction. Mol Aspects Med 2006;27(2-3):95-125.

Tichopad A, Pfaffl MW. Improving quantitative real-time RT-PCR reproducibility by boosting pimer-liked amplification efficiency. Biotechnol Lett 2002;24:2053-7.

Liu W, Saint DA. Validation of a quantitative method for real time PCR kinetics. Biochem Biophys Res Commun 2002;294(2):347-53.

Liu W, Saint DA. A new quantitative method of real time reverse transcription polymerase chain reaction assay based on simulation of polymerase chain reaction kinetics. Anal Biochem 2002;302(1):52-9.

Bewick V, Cheek L, Ball J. Statistics review 14: Logistic regression. Crit Care 2005;9(1):112-8.

Rutledge RG, Stewart D. A kinetic-based sigmoidal model for the polymerase chain reaction and its application to high-capacity absolute quantitative real-time PCR. BMC Biotechnol 2008;8:47.

Westgard JO, Barry PL, Hunt MR, Groth T. A multi-rule Shewhart chart for quality control in clinical chemistry. Clin Chem 1981;27(3):493-501.

Westgard JO, Westgard SA. The quality of laboratory testing today: an assessment of sigma metrics for analytic quality using performance data from proficiency testing surveys and the CLIA criteria for acceptable performance. Am J Clin Pathol 2006;125(3):343-54.

How to Cite
1.
Shahsiah R, Abdollahi A, Azmoudeh Ardalan F, Haghi-Ashtiani MT, Jahanzad I, Nassiri Toosi M. Result Variation and Efficiency Kinetics in Real-Time PCR. Acta Med Iran. (No. 5):279-282.
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Articles