This is a poster presented at the American Association for Aerosol Research, Fall 2010 in Portland, OR. It details some of our work on expression profiling of pathogenic bioaerosols.

1. Hi-Fidelity RNA Recovery for the Expression Profiling of
Airborne Bordetella 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:
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Gene expression patterns can provide a fundamental understanding of how
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airborne microbes respond to atmospheric environmental stresses. To
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obtain in-situ transcription signatures, we present a method that
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successfully isolates RNA from bacterial bioaerosol using calibrated liquid
impingers containing cold Trizol as the sole capture medium. This
approach preserves microbial RNA as it exists in its airborne state,
facilitating the analysis of a true “aerosol expression profile.” Microbial cell
walls are disrupted upon contact with Trizol, which rapidly denatures RNA
and associated proteins. This effectively stops transcription, prevents RNA
degradation, and provides a stable “snapshot” of relative mRNA levels near
the instant of collection. This approach eliminates any artifacts associated
with responses to the physical stresses microbes experience during their
capture in conventional liquids, or on agar and filter surfaces.
From a phosphate buffer containing fetal bovine serum, log phase
SUMMARY OF METHODS:
Bordetella pertussis was aerosolized with a 6-jet Collison nebulizer into a
•Grow 20 agar spread plates from a
well-mixed, 0.8 m3 chamber maintained at 22°C and 85% RH. Bioaerosols
single colony in stages
reached a density > 2 x 1010 microbes/m3, with a mean cell residence time
•Resuspend in 20mL PBS + 1% FBS
between 10 and 40 minutes. Microbes were simultaneously collected in 5
in BGI 6-jet Collison Nebulizer
swirling liquid impingers (SKC biosamplers) containing 20mL Trizol (4°C)
•Humidify chamber to 75% RH
for 40 minutes. Evaporative losses of the aqueous Trizol component
•Nebulize 10min (RH up to 80-85%)
requires volumetric reconstitution with RNAse free water prior to RNA
•Collect aerosol in SKC liquid impingers
extraction. Total RNA yields were well in excess of 3 micrograms - the
containing 20mL Trizol while continuing
minimum mass required for microarray expression analysis.
to nebulize for 30min.
•Monitor and adjust RH in chamber
In conclusion, the use of Trizol as an impinger medium facilitates the
•Stop Nebulizer; continue collection (10 min)
efficient collection and isolation of microbial RNA such that microarray
•Reconstitute Trizol with RNAse free water
expression analysis can be conducted with confidence on bioaerosols
•Extract RNA with standard Trizol protocol
maintained under controlled environmental conditions.
RESULTS: CONCLUSIONS:
Liquid impingers containing Trizol as the sole collection fluid can be used to •Hi-fidelity RNA collected from B. pertussis aerosol has successfully been
collect and “fix” bioaerosol RNA expression profiles with high enough RNA
utilized for expression microarrays, with comparisons to the Pre- and Post-
yields for microarray based gene expression profiling (red line).
aerosolization population in the nebulizer serving as respective controls.
•At the time of printing, these data were being analyzed to identify significant
expression changes in B. pertussis in response to aerosolization.
•Preliminary microarray results suggests a significant change in the
expression of numerous B. pertussis genes in response to aerosolization and
atmospheric environmental stress.
3μg Total RNA •Confirmation of these data by quantitative reverse transcriptase polymerase
chain reaction of specific gene transcripts will follow.