Generation of precise calibration curve for absolute quantification of DNA target by Real-Time PCR. A method for accurate molecular monitoring of t(14;18)-bearing cells in Follicular Lymphoma

Abstract

Objective: Determination of absolute DNA concentration by Real-Time PCR is mandatory for an accurate molecular diagnosis. The critical step is a precise and reliable calibration curve. Plasmid DNA containing cloned target sequences is widely used as standards in quantitative PCR. Usually, the initial molecular number of plasmid molecules is deduced by theoretical calculation and this can be severely limiting the accuracy of PCR assay. Materials and Methods: We have developed a simple method to generate a precise standard curve made of plasmid DNA by competitive PCR approach. The procedure is the following: i) construction of competitor fragment; ii) generation of the stock standard by cloning the DNA target template in the plasmid vector; iii) determination of the number of molecules in the stock standard by competitive PCR; iv) analytical validation of standard curve, in terms of accuracy, sensitivity and specificity. We have used this procedure to quantify the genetic marker breakpoint, the t(14;18), associated to Follicular Lymphoma (FL) of Non-Hodgkin’s Lymphomas (NHL). Results: We found that the assessment of plasmid copy number, obtained by theoretical calculation, is overestimated compared to our competitive PCR approach (error of 22% +/-5). The standard plasmid DNA, used to generate the calibration curve, is stable and produces a high reproducible curve even after a long-term storage compared to genomic DNA curve. Conclusions: The described procedure: i) generates robust standard curves for absolute quantification of any DNA target of interest; ii) is suitable with any molecular diagnostic platform; iii) contributes significantly to improve the accuracy of routine laboratory applications.

Keywords

• molecular diagnostics
• standard curve
• competitive-pcr
• t(14;18)bcl2/igh major breakpoint region