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Resolving Poor PCR assay Efficiency in Real Time PCR

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Information about Resolving Poor PCR assay Efficiency in Real Time PCR
Education

Published on March 9, 2014

Author: ajithnandanam

Source: slideshare.net

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http://technologyinscience.blogspot.com/2013/03/real-time-pcr-inhibition-and-poor.html

Real time PCR efficiency is one of the critical factor, in real time PCR assay development and optimization. A 100% percent efficient PCR result indicates a well optimized assay development. Below which indicates the need to further optimize the reaction parameters, which includes, primer probe concentration, buffer optimization, etc. An accepted PCR assay efficiency ranges from 90% to 110%.

A standard curve can be generated and efficiency can be calculated.
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Real Time PCR: Assay Efficiency www.technologyinscience.blogspot.com

Real Time PCR amplification Plot www.technologyinscience.blogspot.com

Hallmarks of an Optimized PCR Assay • Linear standard curve (R2 > 0.980 or r>|–0.990|) • High amplification efficiency (90–105%). • Consistency across replicate reactions. www.technologyinscience.blogspot.com

Real-time PCR inhibition and poor reaction efficiency • Real time PCR efficiency is one of the critical factor, in real time PCR assay development and optimization. • An accepted PCR assay efficiency ranges from 90% to 105%. A standard curve can be generated and efficiency can be calculated. www.technologyinscience.blogspot.com

Real - time PCR: Causes of high or low efficiency • An assay efficiency above 105% indicates a possible inhibition in the real time PCR reaction. • An assay efficiency below 90% indicates a suboptimized PCR reaction. • One should strive to achieve an assay efficiency of 100% or close to it. www.technologyinscience.blogspot.com

The problem with skewed efficiency in Real time PCR poor efficiency can affect: • assay sensitivity, leading to a smaller dynamic range and decreased versatility. • false conclusions, mainly because targets for comparison will have different efficiencies www.technologyinscience.blogspot.com

How to check the efficiency of real time PCR assay is skewed or not??? • The best method to determine assay efficiency is to generate a standard curve. • A dissociation curve or gel showing multiple peaks or products means there is a competition for reaction resources that almost certainly will have an effect on the reaction efficiency. www.technologyinscience.blogspot.com

Resolution for Poor Efficiency or Inhibition of a real time PCR assay • For inhibition, try to remove the highest concentration and analyse the standard curve, this may bring the assay efficiency close to the desired range. • Re-purifying the template is another option. • Assay optimization is another solution for poor efficiency. It is a laborious process, it can increase the complexity of the assay. www.technologyinscience.blogspot.com

Resolution for Poor Efficiency or Inhibition of a real time PCR assay • Varying Magnesium concentration, from a range of 1.5milli Molar to 6 milli Molar. • Titrating KCl concentration can also improve the assay efficiency. • Primer Probe concentration : Reolve Primer dimer issues Sometimes primer needs to be re-designed for better results. • Try different Annealing Temperatures. (Try Gradient). • PCR additives can also be considered for assay optimization. www.technologyinscience.blogspot.com

PCR Efficiency Calculation: Standard Curve www.technologyinscience.blogspot.com

PCR Efficiency Calculation: • Amplification efficiency denoted by E can be calculated from the below equation: E = 10^(-1/slope); From Standard Curve: y = mx+b; slope (m) = - 3.2746 Amplification Efficiency (E) = 10^(-1 / -3.2746) = 2.02 % Amplification Efficiency = (E-1) * 100% = (2.02 -1)*100% = 102%. Note: A slope value of -3.32 will yield 100% assay efficiency. www.technologyinscience.blogspot.com

Bio-Resource www.technologyinscience.blogspot.com

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