Revisiting the sigmoidal curve fitting applied to quantitative real-time PCR data
Section snippets
Materials and methods
Six gene targets were submitted to qPCR amplification. Three of them (ETS1, PMP22, and TACC1) were amplified in the presence of the SYBR Green dye (B. Dessars et al., in preparation). The PCR amplification of the other three targets (fusion genes E2A–PBX1, mBCR–ABL, and TEL–AML1) was tracked by the fluorescence of a TaqMan probe [12]. All experiments were performed on the 7500 Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, USA), and the results were processed using the SDS
Results
To revisit the SCF methodology, first general concepts and notations are recalled.
Discussion
The widespread use of qPCR and the subsequent high-throughput measurement of gene expression have appealed for some kind of automated algorithm. In this context, the SCF method would be an appropriate choice because it is theoretically able to estimate both relative and absolute gene contents by analyzing the sole amplification curve, thereby avoiding the construction of a standard dilution curve [6], [10]. As for any other method based on an empirical model, the reliability of the results is
Acknowledgments
This study was supported by the Interuniversity Attraction Poles Programme (P6/14), Belgian State, Belgian Science Policy, and by grants from the Fonds de la Recherche Scientifique Médicale and Fonds Erasme to Gilbert Vassart.
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