Abstract
After the introduction of tirosine-kinase inhibitor in chronic myeloid leukemia (CML) treatment, the quantification of the level BCR/ABL positive cells has become essential. Since 1993, our lab has been using a sensitivity qualitative nested RT-PCR assay. Due to the need of tumor cells quantification in CML, we developed, in 2004, a quantitative test by real time PCR. The question was to define whether the relative or absolute quantification was the best strategy. To answer this question our lab team standardized both methods and compared them. The real time PCR (RQ-PCR) was performed in Applied Biosystems® 7500 plataform with TaqMan® probes towards b2a2, b3a2 BCR-ABL transcript and BCR reference gene, in a non multiplex assay. Two separate RQ-PCR reactions were prepared for the BCR-ABL standard and BCR standard. 129 peripheral blood samples of CML patients were tested by both relative and absolute methods. Quality control samples were analyzed in every RQ-PCR run to monitor assay performance: NTC, positive and negative control.
Relative Quantification is based on the expression levels of a target gene (BCR-ABL) versus a reference gene (BCR). This method determines the mRNA level of BCR-ABL gene across mutiple samples and expresses it relative to the levels of an internal control. A pool of c-DNA of 30 patients with untreated CML in chronic phase was used as an internal control (N Engl J Med, 2003, oct 9, 349–15). This method does not require standards with known concentrations and we used Pfaffl mathematical model to calculate the expression of a target gene in relation to a reference gene (Nucl Acid Res.2001; 29:2002–7). The relative expression ratio is calculated from the real time PCR efficiencies and the crossing point of an unknown sample versus a control: Ratio= E(target) ΔCt target (controlsample)/E(reference) ΔCt reference (control-sample)
Absolute Quantification determines the input copy number of the BCR-ABL transcript, usually by relating the PCR signal to a standard curve. The standard curve was constructed using plasmids. Plasmids contaning a cDNA fragment of genes under analysis (b2a2, b3a2 BCR-ABL transcript and BCR gene) were prepared by PCR cloning (Branford S, Br J Haematol, 1999; 107:508–99). A 10-fold diluition series in the range of 106 to 10 copies was prepared for the BCR-ABL transcripts and BCR gene. The results were reported as a ratio of BCR-ABL/BCR % and were expressed relative to the median of BCR-ABL transcripts in the blood of 30 patients with untreated CML in chronic phase (baseline).
Results: We performmed 129 blood samples of CML patients by both the relative and the absolute RQ-PCR. The results showed a positive correlation between relative and absolute values (r = 0.969 and p =0.000).
Conclusion: The results of relative and absolute RQ-PCR methods were equivalent. Although the absolute method is more frequent in literature, the relative quantification presents more simply standardization, low contamination risk since it does not work with plasmids and results as safe as the absolute RQ-PCR.
Development of a Real-Time TaqMan PCR Method for Absolute Quantification of the Biocontrol Agent Esteya vermicola