Understanding the impact of environmental ozone on microarrays
Environmental ozone ([O3]: see Figure 1) can rapidly degrade cyanine 5 (Cy5), the fluorescent dye commonly used to label DNA or RNA in microarray experiments. Cyanine 3 (Cy3) is much less affected by ozone. The degradation of the Cy5 channel in any two-colour microarray experiment will affect theCy5/Cy3 ratio thereby generating unreliable and variable data. The problem has been reported in the literature¹ but, due to its random and intermittent nature, identifying degradation caused by ozone can be challenging. Oxford Gene Technology (OGT) regularly monitor and control ozone levels in our high throughput Genefficiency™ genomic services lab in order to ensure high quality results. This article provides detailed information on the impact of environmental ozone on microarrays and what precautions are taken in OGT’s state-of-the-art Genefficiency genomic services lab to monitor and control ozone.
Figure 1: Ozone consists of 3 oxygen atoms.
What is ozone?
Ozone is an unstable gas occurring in both the upper atmosphere, where it protects the earth from UV radiation and at ground level, where is it considered a pollutant.Ozone is not directly emitted, but is formed by a complex set of reactions involving nitrogen oxides and hydrocarbons in the presence of sunlight. In natural, unpolluted conditions, a cyclic balance is reached where sunlight breaks down nitrogen dioxide to form ozone, which then reacts with nitric oxide to reform nitrogen dioxide. At night, when there is no sunlight to drive the first part of the cycle, ozone is destroyed but not replaced.
Ozone is very reactive which makes it very useful for cleaning and disinfecting. However, it can also corrode building materials, statues and monuments and natural rock features in the landscape. For microarray work, ozone levels as little as 5ppb can affect Cy5 signals and potentially compromise data quality.
It is possible to monitor the local ozone level. For example, the London Air Quality Network² monitors and publishes data for a range of air pollutants including ozone.
Although this type of monitoring gives a good indication as to the local level of ozone, it is much safer to measure the actual ozone level in the laboratory.
Ozone’s effect on microarrays
Ozone can affect the fluorescent signal at different stages during the processing and scanning of microarrays. The first stage where ozone can degrade Cy5 is during the washing process — often referred to as “wet ozone”. Although the slide dries quickly after being taken out of the last wash buffer, the outer edges of each spot will dry before the centre of the spot. This means the edges of the spot become exposed to the atmosphere, and hence to ozone, before the centre. Because ozone is so reactive, the Cy5 signal at the edge of the spot is degraded to a greater extent compared to the signal in the centre of the spot. Ozone degradation cannot always be seen on the tiff image. To really see detail of the spot morphology it is better to look at the profile of the feature, which more clearly shows the intensity of Cy5 across the feature (Figure 2).
Figure 2: Profile view of an array clearly showing reduced Cy5 (red line) intensities across the feature caused by ozone. For less intense features the red signal has completely degraded.
On an array without ozone degradation, the Cy3 (green) and Cy5 (red) profiles should track each other
(Figure 3).
Figure 3: Profile view of an array analysed in the absence of ozone, clearly showing the Cy3(green)and Cy5 (red) profiles tracking each other.
The second stage where ozone can cause a problem is when the slide is dry — often referred to as “dry ozone”. This typically occurs when the slide is being scanned. The effect of ozone is most evident when using a slide holder where a Cy5 degradation gradient can often be seen. This is caused by higher Cy5 degradation at the open end of the slide holder.
Ozone has a relatively short half life of 15 minutes in air. As there is more exchange of air at the open end of the slide holder, ozone levels are refreshed leading to increased degradation of Cy5 compared to the closed end (Figure 4).
Red (Cy5) Channel Only
Green (Cy3) Channel Only
Figure 4: Tiff image of an array that has been scanned in a slide holder without eliminating ozone. The increased air circulation at the open end of the slide holder (left side) causes more ozone to contact the Cy5 thereby causing a visible Cy5 degradation gradient.
Dry ozone can also affect the background as well as the features. Although washing removes most background, some free Cy5 and Cy3 dye will bind to the slide. This Cy5 can also be degraded by ozone and can cause a background gradient to be seen in addition to speckling.
Ozone control in OGT’s Genefficiency high-throughput laboratory
In OGT’s state-of-the-art Genefficiency high-throughput laboratory, ozone levels are accurately measured and carefully controlled. The two stages where ozone can impact microarray analysis, namely processing and washing, are carried out in ozone controlled cabinets. The ozone levels within these cabinets are maintained at 1–4 ppb using NoZone® Ozone Scrubbers. In addition, the ozone levels in the laboratory as a whole are monitored using a Photometric O3 Analyser 400E and logged into our LIMS system. Microarray slides are also stored in ozone controlled cabinets post-use allowing users to refer back to the original slides if required. Through monitoring and controlling ozone levels, Genefficiency customers can be assured of reliable high quality results.
Figure 5: Genefficiency offers state-of-the-art high-throughput sample processing with ozone monitoring and control.
About Genefficiency genomic services
OGT’s proven track record in microarray technology and services — and status as the world’s first Agilent High-Throughput Certified Service Provider — combined with our highly skilled team allows us to provide complete design, project management, and data analysis services to ensure you achieve the highest quality data. We were entrusted with the world’s largest copy number variation study where over 2 billion high quality data points were generated from >20,000 samples in just 20 weeks for the Wellcome Trust Case Control Consortium.
For complete peace-of-mind, trust your project to OGT and benefit from LIMS sample tracking (with over 40 quality checks per sample) coupled with ISO 9001:2008 certification.
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References
- Fare, T. L. et al (2003) Anal. Chem. 75 (17):4672-4675
- The London Air Quality Network, 2010. [online] Available at:http://www.londonair.org.uk/london/asp/default.asp[Accessed 15 June 2010]
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