We propose applications of a PCR-based method for accurate and reproducible transcript level determinations in complex mixtures. This method, kRT-PCR (kinetically-monitored reverse transcription-initiated PCR), supports fully automated quantitation of transcript abundance using total cellular RNA as template and transcript-specific primer pairs. Quantitation derives from a single reaction using a single thermostable DNA polymerase. A computer-controlled digital camera monitors the cyclewise kinetics of PCR product accumulation by fluorescence. Digital image analysis provides for full computer data handling. Transcript copies per cell are computed from the kinetics of PCR product accumulation (directly related to transcript abundance in the cellular RNA template). Preliminary results show that the kRT-PCR assay quantitates transcript level differences between two physiological or genetic states within a factor of 20%. Absolute mRNA levels are quantitated by kRT-PCR assay within a factor of two. Experiments are proposed to further refine the accuracy of kRT-PCR transcript determinations through improved enzymology. Preliminary results show that kRT-PCR assays readily quantitate yeast transcripts ranging from 595 to 0.03 copies per cell. We propose to further assess this assay's detection limits. Approaches are proposed to increase throughput of kRT-PCR transcript determinations by another order of magnitude to quantitate a yeast genome equivalent per analysis. The Experimental Plan includes a number of different applications for the kRT-PCR assay, including: 1. Identification and Quantitation of rare transcripts encoded by known and computationally annotated ORFs. 2. Quantitation of multiple transcripts encoded by a single gene as a consequence of differential RNA splicing and/or multiple promoter elements. 3. Determination of mRNA half lives. 4. Genetic dissection of transcription paths and regulatory networks. 5. Determination of gene copy number in complete cellular genomes. 6. Quantitation of metazoan transcripts using primer pairs specific for known ESTs. The kRT-PCR assay has high accuracy, wide detection range, and flexibility for parallel Quantitation of multiple transcripts in multiple physiological or genetic states as well as cell types. These features enable this assay to address a wide range of genomics-based analyses including, but are not limited to: 1. Quantitation of low abundance transcripts. 2. Analyses of a coordinately expressed, regulated subset of transcripts in a wide varied of cell types or genetic states. 3. Quantitation of cell type specific transcript markers in heterogeneous cell populations (e.g. organs or tissues) as a function of cytokine or therapeutic drug treatment.
