Validation of the 2-^ΔΔCt calculation as an alternate method of data analysis for quantitative PCR of BCR-ABL P210 transcripts

Chronic myelogenous leukemia (CML) is a clonal myeloproliferative disorder that is characterized by the presence of a reciprocal translocation between chromosomes 9 and 22 and results in the formation of the Philadelphia (Phl) chromosome and is present in most of CML patients. The Phl chromosome forms a chimeric gene that encodes an abnormal P210 mRNA transcript in most CML patients. Surveillance for minimal residual disease by detection of BCR/ABL transcripts is currently done mostly by quantitative real-time reverse transcriptase polymerase chain reaction (RT-PCR). Quantitation of BCR/ABL transcripts can monitor tumor load and the outcome of therapy. Absolute quantification determines the input copy number of the transcript of interest, usually by plotting the amount of PCR product onto a standard curve based on serial dilutions of the same product cloned in plasmids. Relative quantification describes the change in expression of the target gene in the patient sample relative to that of a control transcript by using the 2- ^ΔΔCt calculation. The results of real-time RT-PCR for BCR/ABL transcripts are often analyzed by using plasmid DNA standard curves. In the present study, 79 BCR/ABL transcript-positive samples from CML patients who were being monitored for minimal residual disease by real-time quantitative RT-PCR were studied to determine whether the 2-^ΔΔCt approach was equivalent to the plasmid standard curve method. BCR/ ABL P210 transcripts were quantitated using both the plasmid standard curve method and the 2-ΔΔCt calculation. The comparison of both methods revealed a highly significant and linear correlation between the plasmid standard curve method and the 2-ΔΔCt calculation (R² = 0.98, P < 0.0001). Furthermore, there was a reduction of preparation time, contamination risk, and reagent usage. The 2-^ΔΔCt calculation is a convenient alternative method to derive accurate quantitative information from real time PCR assays.