Quantitation of RNA transcripts using genomic DNA as the internal amplification competitor
US6258543A · kind A · utility
Assignee
Inventors
Key dates
| Filing date | Sep 17, 1999 |
| Grant date | Jul 10, 2001 |
| Priority date | — |
| Expiry date | Sep 17, 2019 |
Classification
- Technology area (CPC C)Chemistry; Metallurgy
- CPC primaryC12Q1/6851
- WIPO fieldBiotechnology
- WIPO sectorChemistry
Abstract
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 dete…
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