Slope Efficiency Equals Manageable and Corrected PCR

Posted on Wed, Dec 09, 2009 @ 12:01 PM

A "Referral from the Doctor" Blog Article-  

ε = m.c.2 (shall I explain? slope efficiency equals manageable and corrected PCR)

IF m = manageable dilutions of standard sequence, ideally 10 fold serial dilutions of a plasmid containing known copy numbers of the gene of interest. Copy number of your plasmid control gene is calculated as:

9 x 1012 molecules / Kb = n / μg DNA

Assuming c = corrected data means you have determined the actual copy number in your serial dilutions of plasmid by back calculating from the acquired Ct value, assuming the highest concentration is correct.
AND "2" or ln = is the natural log of a number or based 2 log. As PCR is a function of doubling the copy number with each cycle, all cycles to threshold (Ct) numbers may be compared using a natural log function:

2(delta Ct) or 2(ave Ct of standard 1 - ave Ct of standard 2)

THEN ε = calculated efficiency of the slope would ideally equal 1, meaning 100%

ε =[10(-1/slope)] - 1

Note: When creating standard curves for the purpose of quantitative analysis of real-time RT-PCR, it is essential that the efficiency of the amplification be greater than 88% for all genes analyzed. When making direct comparisons the efficiencies must also be within 5% of each other. The "r" value for each slope must be better than 0.95. A variability of 5% between technical replicates of the standard curve is typical, as is a 10% difference in efficiency between the standard curves of different genes run on the same plate.

Written by: Christina Ferrell, Ph.D., Technical Applications Specialist  

References:
C.A. Heid, J. Stevens, K. J. Livak, P. M. Williams. Real Time Quantitative PCR. Genome Methods (1996) 6:986-994
J. Winer, C.K.S. Jung, I. Shackel, P. M. Williams. Development and validation of real-time quantitative reverse transcriptase-polymerase chain reaction for monitoring gene expression in cardiac myocytes in vitro. Analytical Biochemistry (1999) 270:41-49
R. Higuchi, C. Fockler, G. Dollinger, R. Watson. Kinetic PCR analysis: Real-time monitoring of DNA amplification reactions. Biotechnology (1993) 11(9):1026-1030
K.J. Livak, T.D. Schmittgen. Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT method. Methods (2001), 25: 402 - 408
M.W.Pfaffl. A new mathematical model for relative quantification in real-time RT-PCR. Nucl. Acids Res. (2001) 29:2002-2007
U.E. Gibson, C.A. Heid, P.M. Williams. A novel method for real time quantitative RT-PCR. Genome Res. (1996) 6:995-1001

Topics: Real-time PCR, Referral from the Doctor

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