A method for quantification of absolute amounts of nucleic acids by (RT)–PCR and a new mathematical model for data analysis

Accurate quantification of nucleic acids by competitive (RT)–PCR requires a valid internal standard, a reference for data normalization and an adequate mathematical model for data analysis. We report here an effective procedure for the generation of homologous RNA internal standards and a strategy for synthesizing and using a reference target RNA in quantification of absolute amounts of nucleic acids. Further, a new mathematical model describing the general kinetic features of competitive PCR was developed. The model extends the validity of quantitative competitive (RT)–PCR beyond the exponential phase. The new method eliminates the errors arising from different amplification efficiencies of the co-amplified sequences and from heteroduplex formation in the system. The high accuracy (relative error <2%) is comparable to the recently developed real time detection 5′-nuclease PCR. Also, corresponding computer software has been devised for practical data analysis.