A Method to Compensate for Different Amplification Efficiencies

Abstract

To the Editor-in-Chief: Meijerink et al1 developed a novel efficiency compensation control (ECC) for the quantitation of cells with t(14;18) by real-time polymerase chain reaction (PCR). They showed that a decrease in amplification efficiency in a sample caused an increase in the difference in threshold cycles (Ct) for a multicopy 36-actin gene and a single copy albumin gene, both of which were co-amplified in the ECC reactions. ECC may be applicable as a general control in real-time quantitative PCR using genomic DNA templates. It would have been useful if the authors had described how they quantify t(14;18) in their samples using the calibrator and the ECC. The authors mentioned that the t(14;18) and the 36-actin reactions had equivalent amplification efficiencies, allowing direct normalization of the t(14;18) PCR to the 36-actin PCR at the Ct level. However, PCR efficiencies of independent reactions sometimes differ, making direct normalization inaccurate. They also state that ECC seems useful in identifying patient samples that have PCR inhibitors affecting different reactions to different extents. It would be better to have a way to accurately quantify a target gene in these samples. Since PCR efficiencies may differ between different samples, different tubes, and different primer pairs, one should consider performing PCR reactions on a genomic reference and a standard in the same tube as a target sequence in quantitative PCR. For this purpose, multiplex PCR on a target sequence, a genomic reference, and internal standards for both the target and the reference sequences has been used.2, 3, 4 An internal standard for a target sequence should have the same primer-binding sites as the target sequence but have an internal insertion/deletion or a different probe-binding site to allow its differentiation from the target by size or probe specificity. The target and its internal standard are amplified in the same reaction tube, so that they have approximately equal amplification efficiency. The genomic reference sequence is used to normalize variation caused by initial DNA input and should also have its own internal standard with the same primer-binding sites, so that the reference and its standard have approximately equal PCR efficiency. Four different PCR reactions on the target, the genomic reference, and their respective internal standards in the same tube allow accurate calculation of the target gene dosage, e.g., SMN1 copy number determination for spinal muscular atrophy carrier testing.3, 4 These four reactions can be detected separately by different fluorescence dyes attached to different probes in real-time PCR.