两篇资料分享：
1.A new mathematical model for relative quantification in real-time RT–PCR
Michael W. Pfaffl*
Institute of Physiology, FML-Weihenstephan, Center of Life and Food Sciences, Technical University of Munich,Germany
Received December 18, 2000; Revised February 21, 2001; Accepted March 14, 2001
ABSTRACT Use of the real-time polymerase chain reaction (PCR) to amplify cDNA products reverse transcribed from mRNA is on the way to becoming a routine tool in molecular biology to study low abundance gene expression. Real-time PCR is easy to perform, provides the necessary accuracy and produces reliable as well as rapid quantification results. But accurate quantification of nucleic acids requires a reproducible methodology and an adequate mathematical model for data analysis. This study enters into the particular topics of the relative quantification in real-time RT–PCR of a target gene transcript in comparison to a reference gene transcript. Therefore, a new mathematical model is presented. The relative expression ratio is calculated only from the real-time PCR efficiencies and the crossing point deviation of an unknown sample versus a control. This model needs no calibration curve. Control levels were included in the model to standardise each reaction run with respect to RNA integrity, sample loading and inter-PCR variations. High accuracy and reproducibility (&2.5% variation) were reached in LightCycler PCR using the established mathematical model. INTRODUCTION
2.Standardized determination of real-time PCR ef®ciency from a single reaction set-up
Ales Tichopad, Michael Dilger1, Gerhard Schwarz2 and Michael W. Pfaf¯*
Institute of Physiology and 1Institute of Agronomy and Plant Breeding, FML-Weihenstephan, Center of Life and Food Science, Technical University of Munich, Germany and 2EpiGene GmbH, Biotechnology in Plant Protection,Hohenbachernstrasse 19±21, 85354 Freising, Germany
Received June 24, 2003; Revised July 29, 2003; Accepted August 25, 2003
ABSTRACT We propose a computing method for the estimation of real-time PCR ampli®cation ef®ciency. It is based on a statistic delimitation of the beginning of exponentially behaving observations in real-time PCR kinetics. PCR ground ¯uorescence phase, nonexponential and plateau phase were excluded from the calculation process by separate mathematical algorithms. We validated the method on experimental data on multiple targets obtained on the LightCycler platform. The developed method yields results of higher accuracy than the currently used method of serial dilutions for ampli®cation ef®- ciency estimation. The single reaction set-up estimation is sensitive to differences in starting concentrations of the target sequence in samples. Furthermore, it resists the subjective in¯uence of researchers, and the estimation can therefore be fully instrumentalized.
