Gene expression analysis using total RNA quantities less than one microgram (mu g) is problematic, especially when probing DNA microarrays. Methods to label probe that rely on the polymerase chain reaction can skew the interpretation of mRNA abundance by preferential amplification. The biotechnology industry is witnessing rapid technology breakthroughs for constructing and analyzing microarrays. Ambion sees a need to meet these advancements with complimentary improvements in methods for generating representational-labeled probe. We believe that certain phage RNA polymerases are more flexible than previously thought. We have found support in both the literature and from preliminary experiments at Ambion, that the T3 RNA polymerase can utilize an RNA template. The feasibility of harnessing this activity to efficiently amplify mRNA populations will be determined by this research. The goal of this Phase I proposal is to develop a simple method for representational RNA amplification. We will carry out a comprehensive exploration of a direct mRNA transcription amplification method. The optimal method developed will greatly simplify current antisense RNA (aRNA) amplification techniques, retain the advantages of representational display and will ultimately lend itself to a robust Ambion molecular biology kit. PROPOSED COMMERCIAL APPLICATION: Ambion, a leading supplier of products for RNA purification, quantitation and gene expression analysis, intends to manufacture and market a research kit(s) for direct RNA amplification. This product would provide an improved alternative for researchers needing to amplify and label RNA probes for use on DNA microarrays.