Rosetta Inpharmatics, Inc.

Michael Ziman

Matthew N Ashby

Stewart Scherer

John W Phillips

Nicholas Marini

Colleen P Davis

A random-primed reverse transcriptase-in vitro transcription method of linearly amplifying RNA is provided. According to the methods of the invention, source RNA (or other single-stranded nucleic acid), preferably, mRNA, is converted to double-stranded cDNA using two random primers, one of which comprises a RNA polymerase promoter sequence ('promoter-primer'), to yield a double-stranded cDNA that comprises a RNA polymerase promoter that is recognized by a RNA polymerase. Preferably, the primer for first-strand cDNA synthesis is a promoter-primer and the primer for second-strand cDNA synthesis is not a promoter-primer. The double-stranded cDNA is then transcribed into RNA by the RNA polymerase, optimally in the presence of a reverse transcriptase that is rendered incapable of RNA-dependent DNA polymerase activity during this transcription step. The subject methods produce linearly amplified RNA with little or no 3' bias in the sequences of the nucleic acid population amplified.

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The present invention relates to methods of identifying genes whose expression is indicative of activation of a particular biochemical or metabolic pathway or a common set of biological reactions or functions in a cell ('regulon indicator genes') The present invention provides an example of such an indicator gene. The present invention also relates to methods of partially characterizing a gene of unknown function by determining which biological pathways, reactions or functions its expression is associated with, thereby placing the gene within a functional genetic group or 'regulon'. These partially characterized genes may be used to identify desirable therapeutic targets of biological pathways of interest (&#x201c;regulon target genes&#x201d;) The present invention provides examples of such target genes. Methods for identifying effectors (activators and inhibitors) of regulon target genes are provided. The present invention also provides examples of regulon target gene inhibitors.

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San Francisco, CA, United States of America

Michael Ziman