Published on March 9, 2014
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
Want to watch this again later? Sign in to add this video to a playlist. http://technologyinscience.blogspot.c... Real time PCR efficiency is ...
Real-Time PCR Vs. Traditional PCR ... Agarose Gel resolution is very poor, about 10 fold. Real-Time PCR can detect ... The 5 nuclease assay uses this ...
and use this curve to determine the efficiency of the assay along with R ... poor-quality total RNA ... Real-Time PCR Applications Guide Real-Time ...
Real-time PCR is a form of polymerase chain reaction ... TaqMan® Real-Time PCR Assays; TRIzol™ Reagents; Most Popular Categories. Antibodies & Immunoassays;
Poor PCR Efficiency; ... and 7500 Real-Time PCR System version 2.0 software programs calculate the efficiency of ... perceived lower PCR efficiency of assay:
PCR Protocols & Applications. ... In case of poor PCR efficiency, ... The efficiency of a real-time PCR assay can be calculated by analyzing a template ...
Quantification strategies in real-time PCR 87 ... absolute real-time RT-PCR assay depends on the condition ... If no real-time PCR efficiency assessment ...
• Real Time Pcr at a glance: 38,944 LinkedIn members have this skill. Where they work. Thermo Fisher Scientific (1,328 members) Life Technologies (1,043 ...