Construction and use of a multi-competitor gene for quantitative RT-PCR using existing primer sets

We describe a simple and inexpensive method for the construction of multi-competitor molecules for use as internal standards in quantitative RT-PCR. The construction involves the linking and annealing of 20mer PCR primers with complementary 40mers using either a step-wise or bulk process. The entire construct is then ligated and amplified by PCR prior to cloning. Using this approach, we have constructed a gene containing priming sites for 18 different products of immunological interest, including murine cytokines and cell surface markers, as well as murine beta-actin and T. cruzi rRNA. The cost of production of the competitor is minimized by use of a high-throughput multi-oligonucleotide synthesizer for production of the individual components of the synthetic gene, and by use of the same oligonucleotides in gene construction and as primers for the RT-PCR reactions. This procedure can be applied to the production of other polycompetitor molecules as well as to the construction of other types of synthetic genes.