QUANTITATION OF RNA TRANSCRIPTS USING GENOMIC DNA AS THE INTERNAL AMPLIFICATION COMPETITOR

This invention is directed to methods for the quantitative measurement of specific gene expression levels in biological samples. In one embodiment, methods for the quantitative monitoring of gene expression without either co-amplification of an added template or use of an endogenous constitutive transcript are provided. The former involves a duplex amplification reaction in which a single set of primers is used to amplify both genomic DNA and expressed mRNA from the same gene sequence. These primers are targeted for sequences flanking the splice junction and intron sequences for the mRNA and DNA respectively. By their use, any suitable nucleic acid amplification technology yields mRNA and DNA amplimers which are distinguishable by length and sequence heterogeneity. These amplimers are also present in the final amplification reaction in ratios which are dependent upon the ratios of the expressed mRNA to the DNA in the sample, allowing the quantitation of mRNA in a sample which is normalized to the number of copies of genomic DNA since the genomic DNA acts as the internal quantitation standard, and in effect yields the amount of mRNA per cell. Any detection methodology which can detect amplimers of different lengths or sequences can be used for post amplification quantitation. This strategy may be employed for any gene system in which the mRNA sequence differs from the original genomic DNA sequence. In another embodiment, methods for the quantitative monitoring of gene expression involving determining the ratio of genomic material to expressed mRNA without nucleic acid amplification and/or primer binding are described. This method includes the independent and direct detection of the genomic material and mRNA by complementary probe binding without prior amplification. Additional methods for quantitative measurement of gene expression rates without RNA transcription region introns are described. The invention is useful in gene expression determination in research and commercial applications.