RAPID GENERATION OF HOMOLOGOUS INTERNAL STANDARDS AND EVALUATION OF DATA FOR QUANTITATION OF MESSENGER-RNA BY COMPETITIVE POLYMERASE-CHAIN-REACTION

Sensitive and quantitative measurement of messenger RNA (mRNA) is impo rtant for accurate assessment of gene expression. Conventional methods of mRNA measurement frequently lack the sensitivity required to detec t mRNA expressed at low level, such as mRNA encoding receptors and int racellar signaling molecules. Thus, the extremely sensitive RT-PCR has become the method of choice for examination of gene expression. Howev er, quantitation of mRNA by PCR is difficult because small variations in amplification efficiencies among sample tubes can lead to substanti al differences in product yield, thereby rendering direct comparisons between samples invalid. Development of protocols for quantitative RT- PCR has relied on internal standards to monitor the efficiency of the RT-PCR in different reaction tubes. Technically, the two most serious limitations to routine successful application of competitive quantitat ive PCR is ready access to competitive internal standards and efficien t methods for accurate quantitative analysis of the data. In the prese nt manuscript, application and validation of a simple approach to gene rate homologous internal competitive standards and to quantitate data for rapid, accurate determination of the expression level of genes by quantitative PCR is described. Generation of the competitive standard from a previously amplified PCR product by the methods described requi res only one additional primer pair, and an additional two-step reacti on; it can be completed in 1-2 days. Analyzing the results of the comp etitive PCR reaction via phosphoimager analysis provides a simple, rap id method for accurate quantitation of results. Data presented here cl early illustrate that the methods described have been successfully app lied, and that they should have wide application for competitive quant itative PCR analysis of gene expression. (C) 1998 Elsevier Science Inc .