Thursday, December 15, 2011

Ozone, Dyes & Microarrays

Microarrays analysis uses different types of fluorescent dyes, including: the cyanines family (cyanines 3 and 5), the Alexa Fluor family (Alexa Fluor 555 and 647), the DyLights 547 and 647 or the Oyster 550 and 650. These are all hydrophilic fluorophores used to label DNA, RNA, Proteins and other bio molecules.

As shown in Figure 1, these different fluorescent dyes have a common base structure composed of two aromatic rings, a conjugated carbon chain and two side chains to which the dyes are going to link to the biological molecule of interest.  Conjugation of these dyes to different types of biomolecules is typically done with NHS-esters.

Each dye family includes many molecules different from each other in their side chains (Figure 2 non exhaustive). All of these fluorescent dyes do not have the same applications and some of them can be used in cytology. (1)

The signal emitted from these dyes is result of the double bond conjugation of the carbon chain. Cyanine 3, Alexa 555, Oyster 550 and Dylight 547 are excited at a maxium wavelenght of ≈ 550 nm and emit at ≈ 570 nm, in the green part of the spectrum; and cyanine 5, Alexa 647, Oyster 650 and Dylight 647 excited at ≈ 650 nm, emit in the red part of the spectrum at ≈ 670 nm.
The lateral chain end function is generally composed of N-hydroxysuccinimidyl ester (NHS) or of maleimide. In the case of protein labelling, the maleimide function is preferred because its sulfhydryle group can link to the cystein residues of the interest protein. When a nucleic acid is labeled, cyanines 3 and 5 are preferentially synthetized to carry a NHS-ester reactive group. This function only reacts with aliphatic amines; the nucleotides of interest lack this function so they are modifed with an aminoallyl group. As shown in Figure 3 , cyanine dyes link to terminal 5’ phosphates of the DNA molecule via one of the extremities. (2)

Microarray analysis quality depends on many factors and among the most important are stable ratios of the bound Cy5 and Cy3 dyes. (case of the cyanines dyes family, similar to the other fluorescent dyes). At a concentration of only 5-10 ppb and for duration as short as 30 seconds, cyanine 5, Alexa Fluor 647, DyLight 647 and Oyster 650 are subject to ambient ozone oxidation (Figure 4). Their fast degradation results in an important decrease in fluorescence intensity and consequently in a wrong microarray data interpretation. (3, 4)
Because it is the most frequently used, let’s consider the example of the cyanines family, the other dyes of this document having a comparable reaction. Cyanines 3 and 5 have two similar structures but cyanine 5 is more sensitive to ozone exposure because the ozonolysis reaction (Cy5 oxidation) takes place in the additional carbon-carbon double bond that link the two aromatic rings (Figure 5). Figure 6 shows the general reaction scheme. (5)
Our Ozone Free Work Environment allows to realize hybridizations in an 100% ozone free environment, and thus to keep the cyanines integrity.

Photo of our ozone free work environment with an Agilent microarray scanner installed in it.

This is a photo of our competitors catalyst filters that they use to remove ozone from a work environment. It's about 4 feet long.

This is the size of ours.

For more information about this ozone free work environment please see:

1 W.Chiuman, Y.Li. Efficient signaling platforms built from a small catalytic DNA and doubly labeled fluorogenic substrates. Nucleic Acid Research. pp1-5 (2006)

2 A.Iqbal, S.Arslan, B.Okumus et al. Orientation dependence in fluorescent energy transfer between Cy3 and Cy5 terminally attached to double-stranded nucleic acids PNAS 105 11176-11181 (2008)

3 T.Fare, E.Coffey, D.Hong Yue et al. Effects of atmospheric ozone on microarray data quality. Analytical Chemistry 75, 4672-5 (2003)

4 Invitrogen- Documents presentation by Jakub Razga

5 W.Branham, C.Melvin, T.Han et al. Elimination of laboratory ozone leads to a dramatic improvement in the reproducibility of microarray gene expression measurements. BMC Biotechnology 7:8 (2007)