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.