Originally published : Thu, December 3, 2009 @ 5:09 PM
Updated : Mon, September 19, 2022 @ 2:10 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
