Hi-Fidelity RNA Recovery for the Expression Profiling of Airborne Bordatella pertussis Response to Atmospheric Environmental Stress

KEVIN M. MCCABE (1), Jane Turner (1), Tracy L Nicholson (3), Tod Merkel (2), Mark Hernandez (1)

(1) Civil, Environmental and Architectural Engineering Department, University of Colorado, Boulder, Colorado (2) Laboratory of Respiratory Pathogens, United States Food and Drug Administration, Bethesda, Maryland (3) National Animal Disease Center, United States Department of Agriculture, Ames, Iowa

     Abstract Number: 427
     Last modified: May 12, 2010

Abstract
Examining gene expression changes in airborne microorganisms using microarrays can provide a fundamental understanding of how transcriptionally active bacteria respond to atmospheric environmental stresses. We present a method that successfully isolates RNA from bacterial bioaerosol using calibrated liquid impingers, which contain cold Trizol as the sole capture medium. This approach fixes and preserves microbial RNA as it exists in its airborne state, facilitating the analysis of a true “aerosol expression profile.” Upon contact with the Trizol in an impinger reservior, microbial cell walls are disrupted, and associated RNA and protein structures rapidly denature; this immediately stops transcription, prevents RNA degradation, and provides a stable “snapshot” of relative mRNA levels at the instant of collection. This approach eliminates artifacts associated with capture in traditional liquid media (e.g. water, glycerol or oils) which permit reversion of genetic profiles, as well as on solid-phase filter/membranes where mechanical stresses and desiccation could alter gene expression in ways not directly associated with atmospheric responses. Log phase B. pertussis was continuously aerosolized from a collison 6-jet nebulizer containing 50mM phosphate buffered saline and 1% fetal bovine serum into a 0.8cubic-meter chamber maintained at 85% relative humidity and 22C to an approximate concentration of 2.56X10$^(10) microbes m$^(-3), with an aerosol residence time spanning between 10 and 40minutes. Microbes were simultaneously collected in 5 swirling liquid impingers (SKC biosamplers) containing 20mL Trizol (4C) for 40min; this collection leads to evaporation of the aqueous Trizol component requiring reconstitution with diethylpolycarbonate treated water to the original volume prior to RNA extraction. RNA yields were well in excess of 3 micrograms, the minimum total RNA mass required for microarray expression analysis. In conclusion, the use of Trizol as an impinger medium permits collection and isolation of RNA from aerosolized microorganisms of sufficient quality and quantity to execute microarray expression analysis.