Electronic Letters to:

Endocrine Care: Shuiying Hu, Marge Ewertz, Ralph P. Tufano, Mariana Brait, Andre Lopes Carvalho, Dingxie Liu, Anthony P. Tufaro, Shehzad Basaria, David S. Cooper, David Sidransky, Paul W. Ladenson, and Mingzhao Xing Detection of Serum Deoxyribonucleic Acid Methylation Markers: A Novel Diagnostic Tool for Thyroid Cancer J Clin Endocrinol Metab 2006; 91: 98-104 [Abstract] [Full text] [PDF]

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Is it correct not to apply strategies of modern relative quantification?

Antonio Martinez-Peinado, Javier Caballero-Villarraso, M. Angeles Gálvez-Moreno, and Cristóbal Aguilera-Gámiz   (24 May 2006)

Is it correct not to apply strategies of modern relative quantification? 24 May 2006
Antonio Martinez-Peinado, MD Hospital Reina Sofía. Cordoba, Javier Caballero-Villarraso, M. Angeles Gálvez-Moreno, and Cristóbal Aguilera-Gámiz Send letter to journal: Re: Is it correct not to apply strategies of modern relative quantification? amartinezpeinado{at}auna.com Antonio Martinez-Peinado, et al.	We read with great interest the article by Hu et al. (1). The detection and quantification of circulating DNA, especially methylated forms in peripheral blood, is of great interest in oncology. In the last 3 years some 3,000 articles related to “DNA-Methylation” [MeSH] and “cancer” have been published. Since the beginning of this year some 200 have been published alone. But if the terms “real-time PCR” and “relative quantification” are added to this search, only four papers are found, which may explain the possible mistakes in data interpretation by Hu et al. (1). Although the methodological approximation used by the authors is very interesting, their conclusions are based on data that may be incorrect. Hu (1) proposes a simple ratio “problem gene/reference gene” as a method of measurement. Using Hunt’s tutorial (2), for example, differences of efficiency of two PCR reactions can change the quotients, depending in what point of the curve is used; therefore it is not only a straight line of regression. The mathematical strategies of relative quantification established by Pfaffl (3), Vandesompele et al. (4) or Huggett et al. (5) have clarified how relative quantification must be done. The ideal situation would be to use not only a reference gene for every problem gene but several reference genes, but to never use the same reference gene for several problem genes. On the other hand, not considering the differences of efficiency of PCR reactions can lead to overestimations in some regions of the curves and to underestimations in others. For example, a simple quotient CALCA/β-actin may not have enough robustness using terms of reproducibility to be confident of the results. Therefore it would be necessary to recalculate the ratios correctly before coming to conclusions about the validity of the method. References 1. Hu S, Ewertz M, Tufano RP, Brait B, Carvalho AL, Liu D, Tufaro AP, Basaria S, Cooper DS, Sidransky D, Ladenson PW, Xing M. 2006 Detection of serum deoxyribonucleic acid methylation markers: a novel diagnostic tool for thyroid cancer. J Clin Endocrinol Metab 91:98-104 2. Hunt M. 2004 Real Time PCR Tutorial (http://pathmicro.med.sc.edu/pcr/pcr-home.htm), Univ. of South Carolina Press 3. Pfaffl MW. 2001 A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 29:e45 4. Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A, Speleman F. 2002 Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biology 3:research0034.1 -research0034.11 5. Huggett J, Dheda K, Bustin S, Zumla A 2005 Real-time RT-PCR normalisation; strategies and considerations. Genes Immun 6:279-284