A novel method to compensate for different amplification efficiencies between patient DNA samples in quantitative real-time PCR

Abstract

Quantification of residual disease by real-time polymerase chain reaction (PCR) will become a pivotal tool in the development of patient-directed therapy. In recent years, various protocols to quantify minimal residual disease in leukemia or lymphoma patients have been developed. These assays assume that PCR efficiencies are equal for all samples. Determining t(14;18) and albumin reaction efficiencies for sixteen follicular lymphoma patient samples revealed higher efficiencies for blood samples than for lymph node samples in general. However, within one sample both reactions had equivalent efficiencies. Differences in amplification efficiencies between patient samples (low efficiencies) and the calibrator in quantitative analyses result in the underestimation of residual disease in patient samples whereby the weakest positive patient samples are at highest error. Based on these findings for patient samples, the efficiency compensation control was developed. This control includes two reference reactions in a multiplex setting, specific for the beta-actin and albumin housekeeping genes that are present in a constant ratio within DNA templates. The difference in threshold cycle values for both reference reactions, ie, the Ct(2) value, is dependent on the amplification efficiency, and is used to compensate for efficiency differences between patient samples and the calibrator. The beta-actin reference reaction is also used to normalize for DNA input. Furthermore, the efficiency compensation control facilitates identification of patient samples that are so contaminated with PCR inhibitory compounds that different amplification reactions are affected to a different extent. Accurate quantitation of residual disease in these samples is therefore impossible with the current quantitative real-time PCR protocols. Identification and exclusion of these inadequate samples will be of utmost importance in quantitative retrospective studies, but even more so, in future molecular diagnostic analyses.

Figure 1.

Theoretical schematic illustration of β-actin and albumin amplification curves at high and low reaction efficiencies (>E). The β-actin (black curves) and albumin (gray curves) amplification curves and Ct values at high (solid lines) and low (stippled lines) reaction efficiencies for a hypothetical genomic DNA template concentration. Low reaction efficiencies result in higher Ct values for both reaction in a non-linear relationship (Eq. 1) . DNA templates with higher efficiencies for both reactions will have lower difference in Ct values between both reference reactions (low ΔCt values) than templates with lower reaction efficiencies (high ΔCt values).