An evaluation of competitor type and size for use in the determination of mRNA by competitive PCR

Abstract

The technique of competitive PCR for measuring mRNA is used widely. Several variations of the method have been reported. We have evaluated some of the commonly used competitor types as part of our study into expression of the androgen receptor (AR). These included mutant, intron, deletion construct, and nonhomologous competitors, which were assessed with an emphasis on their ability to amplify the target with the same efficiency, as well as their capacity to form heteroduplexes with it. The effect of competitor size on amplification efficiency was also investigated. We found that the use of a common primer set did not guarantee equal amplification efficiencies among DNAs sharing the same primer sequences. For the competitors evaluated in this study, sequence length was the major determinant of amplification efficiency. The longest competitors were amplified with the least efficiency. Differences in amplification efficiencies were corrected for by standardizing the competitor against the target. Constructing competitors of different sizes to the target may not eliminate heteroduplex formation when they share common sequence with the target as with the intron and deletion type competitors. Such heteroduplexes may interfere with the analysis if they cannot be resolved from both the target and competitor. Use of a mutant competitor constructed by the conversion of one enzyme restriction site to another produced determinations that were independent of both heteroduplex formation and cycle number. A method is described for generating a mutant competitor with a single PCR. The results of this study show that when competitive PCR is being used to determine absolute levels of mRNA, the competitor constructed should be assessed carefully for its ability to amplify with the same efficiency as the target and for its capacity to form heteroduplexes with it.