NuGEN Technologies Inc. has patented a novel means of isothermal linear gene amplification called SPIATM, single primer isothermal amplification. The method has been incorporated into a novel method for isothermal linear RNA amplification (patent pending), termed Ribo-SPIATM. Initial proof-of-concept studies demonstrate that mRNA can be converted to cDNA and amplified from very small total RNA samples. The overall goal of this project is to optimize the Ribo-SPIATM method and demonstrate its utility for gene expression analysis and profiling, in very small number of cells, and ultimately single cells. To achieve this goal, we will demonstrate efficient and highly reproducible global amplification mRNA in very small total RNA samples with maintenance of fidelity of transcript representation. Such amplified nucleic acid will be evaluated using selected transcripts of low and high abundance. Expression levels of selected transcripts will be analyzed using rear-time PCR/Taqman, to validate performance characteristics of the amplification method. In addition, procedures for labeling of the amplification products for analysis of global expression profiles and differential gene expression analysis using microarray technologies will be established. Successful and efficient amplification of nucleic acid from minute samples with maintenance of transcript ratios will have widespread use in the expanding market of gene expression analysis. In particular, the method will enable expression analysis in very small samples such as obtained for need biopsies, laser capture microdissection samples and minute number of cells for analysis of developmental and differentiation processes.
