Micro-array systems utilizing cyanine-5 (Cy5) and cyanine-3 (Cy3) dyes, for comparative genomic hybridization research, have become commonplace in many labs and medical research facilities, throughout the past decade. One of the challenges faced by facilities using this technology is the elimination of environmental factors that may degrade their samples, and interfere with their results.
In a 2003 paper published in Analytical Chemistry Thomas L Fare, and others, showed that ozone levels as low as 5-10 ppb can substantially degrade Cy5 within minutes. This effect has since been the topic of numerous other papers and articles. Some of this research focuses on determining what stages of micro-array testing are most susceptible to ozone degradation. Other research has worked to determine which preventative methods are the most effective at reducing ozone degradation.
The intent of this page is not to further discuss ozone degradation or methods of ozone elimination, and we encourage you to explore the References and Suggested Readings listed below for more on those topics. The intent of this page is to discuss how to monitor ozone levels in a manner appropriate for micro-array applications: ozone levels, detection equipment, and caveats.
Microarray Sample Showing Effect of Ozone*
Ozone-Free Microarray Sample*
To quote one of the conclusions of the paper mentioned above, "...when possible, microarray work should be performed in a laboratory with an HVAC system outfitted with filters that significantly reduce ozone (</= 2 ppb)".
Environmental (ambient) ozone is an issue in almost any lab environment, and it should be of specific concern to any lab located in an urban area. The reason for this is, the outdoor environmental ozone levels are commonly far beyond the threshold mentioned above, and indoor ozone levels will usually track outdoor levels closely, unless special steps are taken to remove ozone from the air.
Environmental ozone levels vary seasonally, but during the "high risk" summer months, it is common to see many US metro areas easily reach 50 - 60 ppb with spikes of up to 100 ppb or more. Indeed, the phenomenon of air pollution drift means that even rural areas experience higher ozone levels than might be expected.
One of the keys to avoiding ozone microarray degradation is proper ozone measurement. Without accurate knowledge of your ozone levels, it is difficult or impossible to determine where the greatest risk of ozone degradation lies within your protocols, and the best preventative measures to suit your facility.
As with anything, proper measurement requires proper equipment. In the world of ozone detection, there are three options:
Much of the literature on the subject of Cy5 ozone degradation refers to their use UV-based equipment, and that is the recommendation of Ozone Solutions as well. Our comparison of ozone detection technologies paints the benefits of UV in broad strokes, but for microarray applications there are a few specific points you should consider:
Read on to find out more...
The OS-4, a popular sensor-based ozone monitor
Despite low-range models with rather tight accuracy ratings, "sensor-based" ozone monitors are designed primarily for human safety purposes. This means that they are targeted for optimal functionality and accuracy around the 0.1 ppm standard set forth by OSHA. At the extremes of their range (including low ppb levels), their accuracy may stray a bit further than stated. Oftentimes, the resolution available is no better than 10 ppb increments, with corresponding accuracy.
Low-range UV ozone analyzers, on the other hand, are designed with precise lab work in mind. Their readings in the low ppb range are a more truthful indication of actual ozone levels.
Since they are based on principles of chemical reactions, sensor-based ozone monitors also run the risk of interference by other compounds that they may encounter in the air. This can lead to cases of false positives, where a random VOC may cause high readings on a monitor despite ozone levels that are, in fact, within range. The simple use of a Sharpie marker near some monitors is enough to make this happen.
The reverse is also true, with some gases inhibiting the responsiveness of ozone monitors. These rare instances could lead to a decrease in ozone response.
UV monitors avoid interference from almost all other gases, due to their principle of UV absorption - a much more selective technology than either electrochemical or semi-conductor based sensors.
Like all other scientific equipment, sensor-based ozone monitors require periodic calibration over the course of their service life. Most manufacturers recommend annual calibration, so most customers will have them checked once every 12 months. Ozone Solutions sells hundreds of these monitors a year, and upon annual service almost all require an upward adjustment in sensitivity to bring them back within calibration.
This trend is an indicator that the ozone response of sensor-based monitors does indeed degrade over time, meaning that reported ozone levels are often lower than actual levels - especially toward the end of a calibration cycle.
Considering their design intent, this decrease in ozone sensitivity is usually acceptable - not exposing users to any undue risk. For microarray applications, however, this decrease in ozone sensitivity could allow increased ozone degradation even when reported ozone levels are within a "safe" range. This results in wasted samples, and worse - time spent trying to determine what other source may be causing degradation when the culprit is hidden behind incorrect ozone readings.
A UV Ozone Analyzer, if properly maintained during the course of a calibration cycle (regular replacement of intake filters, occasional zeroing with a zero-cartridge), will maintain a fairly tight accuracy rating throughout that time. In addition, the diagnostic circuitry that is present on this class of equipment will often alert the user to failing conditions before they become a problem.
Ozone Solutions recommends UV-based ozone analyzers for any application where microarray work is being performed. UV Ozone Analyzer technology allows for accuracy at low ppb levels, resistance to interference and reliable performance throughout their calibration cycle.
Click through to our UV-based ozone analyzers product page to learn more about UV analyzers and the benefits they provide to microarray applications. Specifically, the API-465L and UV-106L (pictured below) are both excellent choices for microarray applications due to their fine-grained resolution and ability to read reliably at low-ppb ozone concentrations.
A sampling of UV Ozone Analyzers
The API-465L (Left) and the UV-106L (Right) are both good choices for low-ppb level ozone measurements.
Authors: Thomas L. Fare, Ernest M. Coffey, Hongyue Dai, Yudong D. He, Deborah A. Kessler, Kristopher A. Kilian, John E. Koch, Eric LeProust, Matthew J. Marton, Michael R. Meyer, Roland B. Stoughton, George Y. Tokiwa, and Yanqun Wang
(Rosetta Inpharmatics LLC, 12040 115th Avenue NE, Kirkland, Washington 98034,
Agilent Technologies, 395 Page Mill Rd3500 Deer Creek Road, Palo Alto, California 943034)
Analytical Chemistry 10.1021/ac034241b
Abstract: A data anomaly was observed that affected the uniformity and reproducibility of fluorescent signal across DNA microarrays. Results from experimental sets designed to identify potential causes (from microarray production to array scanning) indicated that the anomaly was linked to a batch process; further work allowed us to localize the effect to the posthybridization array stringency washes. Ozone levels were monitored and highly correlated with the batch effect. Controlled exposures of microarrays to ozone confirmed this factor as the root cause, and we present data that show susceptibility of a class of cyanine dyes (e.g., Cy5, Alexa 647) to ozone levels as low as 5-10 ppb for periods as short as 10-30 s. Other cyanine dyes (e.g., Cy3, Alexa 555) were not significantly affected until higher ozone levels (>100 ppb). To address this environmental effect, laboratory ozone levels should be kept below 2 ppb (e.g., with filters in HVAC) to achieve high quality microarray data.
Authors: Steve Byerly, Kyle Sundin, Rajiv Raja, Jim Stanchfield, Bassem A. Bejjani, and Lisa G. Shaffer
From the Signature Genomic Laboratories, Spokane, Washington; and SciGene, Sunnyvale, California
Journal of Molecular Diagnostics, Vol. 11, No. 6, November 2009
Abstract: The increasing prevalence of array-based compara- tive genomic hybridization in the clinical laboratory necessitates the implementation of quality control measures to attain accurate results with a high level of confidence. Environmental ozone is present in all industrialized cities and has been found to be detri- mental to array data even at levels considered accept- able by US Environmental Protection Agency stan- dards. In this study, we characterized the effect of ozone on microarray data on three different labeling platforms that use different fluorescent dyes (Cy3 and Cy5, Alexa Fluor 555 and Alexa Fluor 647, and Alexa Fluor 3 and Alexa Fluor 5) that are commonly used in array-based comparative genomic hybridization. We investigated the effects of ozone on microarray data by washing the array in variable ozone environments. In addition , we observed the effects of prolonged expo- sure to ozone on the microarray after washing in an ozone-free environment. Our results demonstrate the necessity of minimizing ozone exposure when wash- ing and drying the microarray. We also found that washed microarrays produce the best results when immediately scanned; however, if a low-ozone envi- ronment is maintained, there will be little compromise in the data collected.
Authors: William S Branham, Cathy D Melvin, Tao Han, Varsha G Desai, Carrie L Moland, Adam T Scully and James C Fuscoe
BMC Biotechnology 2007, 7:8 doi:10.1186/1472-6750-7-8
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 approximately 22 ppb to 46 ppb and can be as high as 60 to 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 approximately 2 to 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 to 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.
Ozone and Cy5: http://dunham.gs.washington.edu/ozone.html
Understanding the impact of environmental ozone on microarrays: http://www.ogt.co.uk/resources/literature/437_understanding_the_impact_of_environmental_ozone_on_microarrays
Ozone, Dyes & Microarrays: http://arrayit.blogspot.com/2011/12/ozone-microarray.html
Emissions Drift: http://www.noaanews.noaa.gov/stories2010/20100120_ozone.html
* Image Credit:
Microarray images used courtesy of Maitreya Dunham, PhD (University of Washington Department of Genome Sciences). Dr. Dunham has graciously allowed use of images from her website (cited above). Use of these images does not constitute product endorsement.
Last Updated: July 28, 2014