Identifying ozone damage on your arrays
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 result in an incorrect Cy5/Cy3 ratio thereby generating unreliable 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 regularly monitor and control ozone levels in our high throughput Genefficiency™ Genomic Services labs in order to ensure high quality results. However, there are a number of preventative measures that can be taken in any lab to prevent ozone damage affecting array data. This article provides a brief overview of the effect of ozone on microarray data and ways in which ozone can be eliminated.
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. There are many ozone monitoring options available. At OGT we have a large static ozone analyser and also a small portable unit.
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 air³. 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.
Preventing ozone-induced degradation
If the ozone levels in the laboratory are above 5ppb, there are several options to prevent ozone-induced degradation.
NoZone® Workspaces
NoZone Workspaces (Figure 5) consist of a bench top polycarbonate enclosure with an external ozone filtration system. Inside the Workspace, ozone levels are maintained as low as 5ppb within a few minutes of closing the door. This is an ideal area in which to situate the scanner, the washing dishes or, as shown in Figure 5, the LittleDipper® array processor. In addition, slides can be placed inside the Workspace for long-term storage. A key advantage of these Workspaces is the fast and complete depletion of ozone, while the absence of solvents — as used in other ozone depletion methods — ensures no additional waste disposal requirements.
Figure 5: The Scigene NoZone WS Workspace, available in Europe from OGT, contains an ozone filtration system to create an ozone-free work space for array washing and scanning.
Agilent’s Stabilisation and Drying Solution
Agilent’s Stabilisation and Drying Solution is a near-saturated solution of an ozone scavenger dissolved in acetonitrile. This solution is used as a final wash before scanning the array and will prevent both wet and dry forms of ozone degradation. Use of this solution will preserve the dye intensity of the array for several months⁴. As a solvent, strict acetonitrile disposal regulations may apply.
Agilent’s Ozone-Barrier Slide
To prevent the dry mode of ozone degradation during scanning (using an Agilent scanner), it is possible to use a barrier slide which sits over the microarray. This seals the array in a small chamber and limits the extent of ozone degradation5. Barrier slides are effective at preventing ozone degradation during scanning; however, the will not adequately prevent the slide from damage during long-term storage.
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]
- Eco Sensors, Inc., 2010 [Online] Available at: http://www.ecosensors.com/MO-101.pdf [Accessed 15 June 2010]
- Agilent Technologies, Inc., 2010 Development of a Dye-Protecting Solution for Preventing Fluorescent Dye Degradation on DNA microarrays [online]. Available at:http://www.chem.agilent.com/Library/posters/Public/5989_0924en_ozone_abrf.pdf [Accessed 15 June 2010]
- Agilent Technologies, Inc., 2010 Agilent Ozone-Barrier Slide Cover User Guide [online]. Available at:http://www.chem.agilent.com/Library/usermanuals/Public/G2505-90050_OzoneBarrier_Usr.pdf [Accessed 15 June 2010]
Ordering information
NoZone WS Workspaces (various sizes available)
Stabilisation and Drying Solution (p/n 5185-5979): www.agilent.com
Ozone-Barrier Slide (p/n G2505-60550): www.agilent.com
Find out more
For more information about any OGT product or service, please contact us.