Quantitative Real-Time Polymerase Chain Reaction: Methodical Analysis and Mathematical Model
Stefan Wilkeninga and Augustinus Baderb
Research Centre for Biotechnology, Braunschweig, Germany; bBiomedicalBiotechnological Center, Leipzig, Germany
two transcription variants of the glutathione S-transferase 1 gene,with over 100 bp length difference, could not be distinguished by this method. Furthermore, an equation was set up describing the correlation between polymerase chain reaction efficiency and crossing point.This equation can be used to estimate the number of template molecules without having a standard of known concentration. Finally, experimental reproducibility of the real-time polymerase chainreaction was defined. KEYWORDS: Quantitative real-time polymerase chain reaction, LightCycler, melting curve analysis, polymerase chain reaction efficiency, cDNA stability.
Real-time polymerase chain reaction was established for 16 genes using the LightCycler system to evaluate gene expression in human hepatocytes. During the experiments a large set of data has been obtained. These data have nowbeen evaluated with respect to template stability, accuracy of melting curve analysis, and reproducibility. In addition, the statistical evaluation of the efficiencies of all 16 polymerase chain reactions led to a new mathematical model.To examine template stability, the degradation of mRNA and cDNA was determined at different temperatures. Surprisingly, cDNA, which was obtained by first-strandsynthesis, appeared to degrade significantly faster than the respective mRNA. Melting curve analysis is a fast and sensitive method to check for polymerase chain reaction specificity. However, we show that
ADDRESS CORRESPONDENCE AND REPRINT REQUESTS TO: Stefan Wilkening, German Cancer Research Center (DKFZ), Molecular Genetic Epidemiology, Im Neuenheimer Feld 580, 69120 Heidelberg, Germany(tel.: 49-6221-421803; fax: 49-6221-421810; email: firstname.lastname@example.org). This work was supported by a fellowship from the Dr. HilmerStiftung for Stefan Wilkening.
n a previous report, we described the use of realtime polymerase chain reaction (RT-PCR) to quantify a large number of genes coding for phase I and phase II enzymes of drug metabolism.1 This was done with human hepatocytes using theLightCycler system and SYBR green I as fluorescent marker. In the current work we analyzed single experimental steps of this method. The key point for quantitative RT-PCR is the quality of the template for reverse transcription (mRNA) and the template for the PCR (cDNA). To get a general idea of the stability of these templates, we determined mRNA and cDNA degradation at different storagetemperatures. Thereby we could disprove the assumption of many scientists that DNA in general is more stable than RNA. Furthermore, we demonstrate the limits of melting curve analysis in the LightCycler, showing that two significantly different PCR amplificates are indistinguishable by this method. For an absolute quantification by RT-PCR, standard curves are normally generated with standards of knownconcentration. Presupposing that the number of PCR products at the crossing point (CP) is always similar, it is possible to estimate the original number of template molecules only by determining the PCR efficiency. To do so, we set up an equation. Additionally, we determined the reproducibility of RT-PCR with the LightCycler by specifying intra- and interassay variations.
JOURNAL OF BIOMOLECULARTECHNIQUES, VOLUME 15, ISSUE 2, JUNE 2004
S.WILKENING AND A. BADER
Temperature-dependent degradation of mRNA and cDNA. values are given as percent of the molecule number at day 0 (mean ± SD).Total RNA samples for this experiment were isolated from two different cultures of human hepatocytes and were examined for the expression of two genes (glyceraldehyde-3-phosphate...