Methodology article

Elimination of laboratory ozone leads to a dramatic improvement in the reproducibility of microarray gene expression measurements

William S Branham¹ , Cathy D Melvin² , Tao Han¹ , Varsha G Desai¹ , Carrie L Moland¹ , Adam T Scully³ and James C Fuscoe¹

¹  Center for Functional Genomics, Division of Systems Toxicology, National Center for Toxicological Research, U. S. Food and Drug Administration, 3900 NCTR Road, HFT-130, Jefferson, Arkansas 72079, USA

²  Arkansas Regional Laboratory, Office of Regulatory Affairs, U. S. Food and Drug Administration, 3900 NCTR Road, SWR-570, Jefferson, Arkansas 72079, USA

³  Facilities Design and Construction, National Center for Toxicological Research, U. S. Food and Drug Administration, 3900 NCTR Road, HFT-130, Jefferson, Arkansas 72079, USA

author email corresponding author email

BMC Biotechnology 2007, 7:8doi:10.1186/1472-6750-7-8

Published:	12 February 2007

Abstract

Background

Environmental ozone can rapidly degrade cyanine 5 (Cy5), a fluorescent dye commonly used in microarray gene expression studies. Cyanine 3 (Cy3) is much less affected by atmospheric ozone. Degradation of the Cy5 signal relative to the Cy3 signal in 2-color microarrays will adversely reduce the Cy5/Cy3 ratio resulting in unreliable microarray data.

Results

Ozone in central Arkansas typically ranges between ~22 ppb to ~46 ppb and can be as high as 60–100 ppb depending upon season, meteorological conditions, and time of day. These levels of ozone are common in many areas of the country during the summer. A carbon filter was installed in the laboratory air handling system to reduce ozone levels in the microarray laboratory. In addition, the airflow was balanced to prevent non-filtered air from entering the laboratory. These modifications reduced the ozone within the microarray laboratory to ~2–4 ppb. Data presented here document reductions in Cy5 signal on both in-house produced microarrays and commercial microarrays as a result of exposure to unfiltered air. Comparisons of identically hybridized microarrays exposed to either carbon-filtered or unfiltered air demonstrated the protective effect of carbon-filtration on microarray data as indicated by Cy5 and Cy3 intensities. LOWESS normalization of the data was not able to completely overcome the effect of ozone-induced reduction of Cy5 signal. Experiments were also conducted to examine the effects of high humidity on microarray quality. Modest, but significant, increases in Cy5 and Cy3 signal intensities were observed after 2 or 4 hours at 98–99% humidity compared to 42% humidity.

Conclusion

Simple installation of carbon filters in the laboratory air handling system resulted in low and consistent ozone levels. This allowed the accurate determination of gene expression by microarray using Cy5 and Cy3 fluorescent dyes.