Ozone Contamination

The quality of microarray work can be impacted by an environmental contaminant - i.e. ozone. Ground-level ozone can affect lung health, corrode buildings and interferes with the processing of microarrays. This form of ozone is formed by a complex series of chemical reactions between nitrogen oxides and hydrocarbons, mainly from car exhausts, in the presence of sunlight.

Microarray analysis relies upon the binding of two fluorescent dyes - cyanine5 (Cy5) which is red and cyanine3 (Cy3) to test and reference samples respectively. The samples are exposed to the microarray and its thousands of gene probes which are fixed to its surface. Exposure leads, on processing, to a pattern of red, green and yellow dots which can be analysed to discover the differences in gene expression between test and sample tissue (most often diseased and healthy tissue). The pattern contains a mass of research and clinical information, but only if the ratio between the intensity of the Cy5 and Cy3 fluorescent signals is accurate.

Ideally, Cy5 and Cy3 would react only with their respective samples. Unfortunately, this is not the case. Ozone is a very reactive molecule (that is why it causes so much health damage in the lungs). It is known to react more with Cy5 than with Cy3. This happens with ozone levels as low as 5-10 ppb (parts per billion, which are not uncommon in large cities on a daily basis and where most of the key microarray research labs are located). Therefore, in the presence of indoor ozone pollution (which is not routinely measured but is related to outdoor ozone pollution), the Cy5/Cy3 ratio signal cannot be relied on, rendering data from microarray experiments (which are expensive and often paid for from public funds) unreliable and maybe even useless.

Fortunately, researchers and instrument companies are onto the ozone pollution problem with microarrays and have come up with a number of solutions. Ozone damage to Cy5 occurs at two stages in microarray processing - namely, during washing of the slide and when the slide is being scanned (this is when exposure to atmospheric ozone is greatest). The first step is to install an ozone monitor and be aware that damage to data will occur when levels are greater than 5 ppb. Then, the solutions are:

• Enclose the workspace for washing and the scanner with a polycarbonate enclosure that is fitted with an ozone filtration system that can maintain ozone levels inside to 5 ppb or less.
• Use a concentrated solution of ozone scavenging chemical as a final wash before scanning.
• Use a barrier slide to cover the microarray. This will help protect the slide during the scanning process.

It could be wise to employ all of these approaches to optimise the processing of microarrays in the presence of ozone. After all, such advanced technologies deserve the best solutions in order to extract the highest quality data, particularly if it is to contribute to human medical research.