| abstract |
The present invention relates to methods and kits for amplification of mRNA using a primer in PCR that contains an RNA polymerase promoter. The invention provides methods for amplification and detection of RNA derived from a population of cells, preferably eukaryotic cells and most preferably mammalian cells, which methods preserve fidelity with respect to sequence and transcript representation, and additionally enable amplification of extremely small amounts of mRNA, such as might be obtained from 106 mammalian cells. In typical embodiments of the invention, an RNA polymerase promoter (RNAP) is incorporated into ds cDNA by priming cDNA amplification by polymerase chain reaction (PCR) with an RNAP-containing primer. Following less than 20 cycles of PCR, the resultant RNAP-containing ds cDNA is transcribed into RNA using an RNA polymerase capable of binding to the RNAP introduced during cDNA synthesis. This combination of PCR and in vitro transcription (IVT) enables the generation of a relatively large amount of RNA from a small starting number of cells without loss of fidelity. RNAs generated using this method may be labeled and employed to profile gene expression in different populations of cells, e.g., by use of a polynucleotide microarray. |