Amplifying mRNAs for Bacterial Array Analysis

DESCRIPTION (provided by applicant): The revolution in microbial genome sequencing will have a major impact on our understanding, prevention, and treatment of infectious diseases. The advent of DNA array technology has made it possible to evaluate complete gene expression patterns for bacterial pathogens under a variety of experimental conditions. DNA arrays have already enabled analyses of the gene expression response of host cells to interactions with pathogenic organisms. However, robust and reproducible methods are not currently available for measuring global gene expression responses of bacteria during interactions with host cells and during actual infections. This capability was recently referred to as the "Holy Grail" of pathogen expression analysis. Technical limitations of currently available methods for purifying bacterial mRNAs have prevented these types of studies. Our long term goal then is to develop and enable techniques that allow scientists to utilize powerful DNA array technology for quantifying bacterial gene expression during microbe-eukaryotic host cell interactions. Our Specific Aims are 1. Enable detection, with DNA arrays, of bacterial gene expression using bacterial mRNA purified from complex mixtures of bacterial and eukaryotic host cell RNA a. Optimize removal of excess rRNAs (prokaryotic & eukaryotic) from complex total RNA mixtures and minimize loss of bacterial mRNAs b. Utilize novel procedures developed in a. and demonstrate detection of previously undetectable bacterial transcripts with DNA arrays 2. Enable detection, with DNA arrays, of bacterial gene expression from limiting amounts of RNA a. Develop/evaluate novel technologies for the linear amplification of bacterial mRNA populations b. Amplify bacterial mRNA from limiting amounts of starting material and demonstrate detection of previously undetectable transcripts with DNA arrays