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The Flexible matriXarray Chip System: A New Perspective,,,for Probe Optimization in Gene-Expression Profiling

In general, oligonucleotide microarrays are composed of a two- or three-dimensional solid surface where DNA, used as a capture molecule (probe), is attached in an orderly fashion. Each of the oligonucleotide probes on the microarray corresponds to a specific target gene, allowing the researcher to hybridize and detect multiple sequences in parallel. The synthesis principle of the oligonucleotides varies among different microarray platforms. Probe sequences can be synthesized ex situ and may then be robotically spotted onto the surface, or probe sequences can be synthesized in situ on the surface of the array.

The Virtual Flask Technology

CombiMatrix Corporation has developed a proprietary electrochemical on-chip synthesis principle. The oligonucleotide probes are synthesized in situ on a special reaction matrix above the semiconductor-based supportive material. A special buffer system allows the generation of so-called virtual flasks in which the oligonucleotide synthesis takes place. Each feature (electrode) on the array is individually addressable and thus allows the fast production of a customized chip (Figure 1).

The matriXarray Chip System from Roche Applied Science is built upon this virtual flask technology (Figure 2). Arrays are ordered online, produced in a quality-controlled environment, and delivered within a bar codelabeled protective flow cell. The HybReader Instrument, a walk-away device for array hybridization and imaging; the corresponding buffer sets; and a software suite for image processing, data extraction, and data mining, complete the integrated matriXarray Chip System.

Probe Optimization Is Crucial

The importance of the flexible production of customized arrays becomes obvious when different probes that detect the same gene are analyzed. Probe design software solutions are not yet able to provide probes with identical thermodynamic behavior. Furthermore, the in silico check for probe specificity is largely related to the transcriptome analyzed, as well as to the amount of sequence information available. Even for the human genome, this information is far from being complete. Currently, microarray vendors are addressing this problem with two different approaches. In the first approach, one probe per gene is offered with in silico and sometimes experimental validation. In the other approach, different probes per gene are used together with platformspecific algorithms to calculate the result. In conclusion, the optimization of probes used in array experiments is of major importance, especially when questions concerning genome-wide screening of gene-expression profiles must be answered.

New Concept for Probe Optimization

Roche Applied Science has developed a new concept for probe optimization using the matriXarray Chip System. The systematic approach analyzes probe profiles and is based on the assumption that specific probes show similar signal-intensity patterns in different samples (e.g., tissues). Preliminary experiments in three different rat tissues demonstrate the feasibility of this approach: a total of 57 different genes are included in the analysis (15 specific for kidney, 1 specific for kidney and liver, 18 specific for liver, and 23 specific for brain) together with 11 housekeeping genes. The chips were hybridized in duplicate. The mean average signal intensity after local backgro und correction and normalization between the tissues via housekeeping genes was calculated. To demonstrate the approach, the resulting probe profiles of one gene for each tissue are displayed in Figure 3.

The comparison of probe profiles reveals the similar behavior of some probes, while others show distinct differences. For the brain-specific gene (GenBank acc. no. AB049056), all five probes show the same tendency; however, probes 3 and 4 display the most preferred pattern due to their high degree of profile similarity and specificity for the brain tissue. Representing the liver-specific gene (GenBank acc. no. J02585), probes 2, 5, and 6 display similar attributes; however, especially probes 4 and 7 show a more distinct pattern (a high signal in brain and/or kidney and a lower signal in liver compared to the other probes), indicating a dysfunction of these two probes. For the kidney- specific gene (GenBank acc. no. AF062389), probes 2, 5, 6, and 7 seem to be specific, while probes 1, 3, and 4 show aberrations in their intensity pattern.

In an ongoing study, the expression levels of 130 genes in 5 different human tissues are analyzed. The goal of this study is to establish an automated workflow for probe selection.

Fast and Convenient Iteration of Chip Layout

In conclusion, the data shown here demonstrate the impact of experimental probe validation in gene-expression profiling experiments. Based on the high flexibility in the production of customized chips, the matriXarray Chip System is the first platform that allows a fast and convenient iteration of chip layouts. Since probe optimization is crucial for all microarray applications, future work will extend this approach to applications outside of ex pression profiling, such as mutation detection and the analysis of splice variants.



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