Agilent (Palo Alto, CA) rely on the in situ synthesis of probes at or near the surface of the microarray slide by ink jet printing using phosphoramidite chemistry.
Agilent oligonucleotide microarrays consist of 60-mers contrasting with the short 25-mers probes employed by Affymetrix. Although short oligonucleotides should in theory provide the greatest discrimination between related sequences, they can often have poor hybridization properties. Linkers have been used to extend the oligonucleotide away from the surface of the slide in certain microarray designs. Agilent however has utilized the actual probe sequence as the linker to extend the probe and thereby provide greater specificity.
Hughes et al., (2001) examined the effects of hybridization specificity and concluded that the beneficial effects of long oligonucleotides are due to both steric and non-steric effects. The importance of a particular base to hybridization efficiency was observed to be roughly proportional to its distance from the surface, suggesting that the benefit of the additional length is partly due to displacement of the 5' end from the surface. Sequence information near the surface anchored 3' end was also found to be relevant as mismatched oligonucleotides hybridized inefficiently compared to perfectly matched oligonucleotides.
The 60-mer format provides enhancements in sensitivity over the 25-mer format partly due to the larger area available for hybridization. A major advantage of Agilent oligonucleotide microarrays is that they require only one 60-mer per gene or transcript. The longer 60-mers are also more tolerant of sequence mismatches, and are thus are more suitable for the analysis of highly polymorphic regions.
The standard experimental paradigm compares mRNA abundance in two different biological samples, either on the same microarray or different microarrays. Agilent oligonucleotide microarrays like conventional spotted microarrays can be used in a two-color scheme design, where the same array is hybridized with two different samples. Two-color experiments are competitive hybridizations with two different fluorescent samples, in which gene expression levels are commonly defined in ratiometric terms. Essentially, they compare transcript abundance between two different biological samples, on the same microarray. One fluorescent target is prepared from a reference mRNA and the second from mRNA isolated from the treated cells or a disease tissue under investigation. The one color design is similar to the Illumina or Affymetrix platforms, where each sample is hybridized to a single chip. This approach although more costly has the advantage of avoiding potential dye bias that can be introduced in the two color experimental design.
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Hughes T.R., Mao M., Jones A.R., Burchard J., Marton M.J., Shannon K.W., Lefkowitz S.M., Ziman M., Schelter J.M., Meyer M.R., Kobayashi S., Davis C., Dai H., He Y.D., Stephaniants S.B., Cavet G., Walker W.L., West A., Coffey E., Shoemaker D.D., Stoughton R., Blanchard A.P., Friend S.H., Linsley, P.S. Expression profiling using microarrays fabricated by an ink-jet oligonucleotide synthesizer. Nat. Biotech, 19 342-347 (2001).
Last Updated November 2012
By Dr. Gary Hardiman