Robotically spotted microarrays

Robotically spotted microarrays (figure 3.2), namely, the robotically spotted cDNA glass slide, were introduced into common use at Stanford University and first described by Mark Schena et al. in 1995 [157]. They are also known as cDNA microarrays.

Figure 3.2: Robotically spotted microarray hybridized to two samples, each stained with two colored dyes. An overview of procedures for preparing and analyzing cDNA microarrays and tumor tissue. Reference RNA and tumor RNA are labeled by reverse transcription with different fluorescent dyes (green for the reference cells and red for the tumor cells) and hybridized to a cDNA microarray containing robotically printed cDNA clones. The slides are scanned with a confocal laser scanning microscope, and color images are generated for each hybridization with RNA from the tumor and reference cells. Genes upregulated in the tumors appear red, whereas those with decreased expression appear green. Genes with similar levels of expression in the two samples appear yellow. Genes of interest are selected on the basis of the differences in the level of expression by known tumor classes (e.g., BRCA1 mutation-positive and BRCA2 mutation-positive). Bioinformatics analysis determines whether these differences in gene expression profiles are greater than would be expected by chance. (Derived from Hedenfalk et al. [90].)

In making the array, a robotic spotter mechanically picks up specific cDNA sequences, typically amplified from vectors in bacterial clones using PCR, from separate physical containers and deposits them in specific locations in the grid on the glass slide to create specific probes. Each cDNA drop should ideally be equal in quantity. This fabrication approach epitomizes the do-it-yourself tendency in microarray measurement, even though there are several commercial ready-to-use versions available. The frequently home-grown quality of these arrays has led to to the production of highly localized and customized microarrays which pose specific challenges in the dual tasks of background noise reduction and foreground RNA signal amplification during subsequent data analysis stages. These challenges are discussed in section 3.2. Designer-definable parameters exist to control spot-basing size, the amount of time for drying, as well as experimental parameters concerning the glass slide material to be used.

There are several advantages and disadvantages in using a robotically spotted microarray:

• The first advantage is customizability. A large subsequence (~ 2 x 103 base pairs long) complementary to the actual sequence that is to be probed is laid down on the chip by the designer who has full control over the species of probes that are to be used. This means, e.g., that if one wishes to make customized chips for a specific purpose, such as for probing the expression of specific RNA in certain cell types, or specific polymorphisms of those genes, one can design a layout and can direct a robotic spotter to make these microarrays. The setup cost of this approach is on the order of $20,000 and detailed guidelines may be found at the Brown laboratory microarraying website.[2] Additionally, companies such as Affymetrix, Amersham Pharmacia, BioRobotics, and Cartesian Technologies sell robotic spotters-arrayer units. Glass slides are available from a variety of vendors specifically for microarray construction. Commercial aspects of the manufacture of such chips appear to be expanding. The notable disadvantage with greater customizability is that it may lead to more possibilities for errors. For instance, poor quality control or nonuniformity in the construction of different species probes such as spot-basing size will complicate the subsequent analysis and interpretation of the resulting chip data.

• The second advantage of robotically spotted microarrays is that larger pieces, or entire cDNAs, are placed on the chip, thus reducing the likelihood of nonspecific hybridization of labeled sample probes to the probe that was laid on the chip. Typically, a designer who wishes to have a particular gene probe on a chip will create a clone with the 52 and 32 ends of that particular gene of interest. The substrings of these cDNAs of the original gene are on the order of 100 to 200 base pairs long. A distinct weakness here is that even though a long probing subsequence ensures a sufficiently confident representative substring of the original gene, it does not mean that hybridization conditions will be fully and equally optimized for all species of cDNA subsequences. As we have noted earlier, the probe-sample probe hybridization rate is known to vary depending upon the GC content of a transcript.

• The third advantage is that RNA from two different samples (typically a test and a control condition) can be hybridized onto a common cDNA microarray substrate at the same time. The two separate RNA samples are typically labeled with different fluorescentdyes, such as Cy3 and Cy5. The two-dye system allows for the excitation of the microarray by laser light at two different frequencies and thus the image of hybridized RNA abundance can be scanned for both colors (corresponding to distinct samples) separately. Since the hybridization conditions, and thus the brightness, of any one spot is not the same as another spot, the individual signals are not typically used separately. Instead, the calculated signal is the ratio or fold difference in the brightness of the hybridized RNA of one sample versus another, specifically the intensity of Cy3 versus Cy5, i.e., Cy3/Cy5. If a background intensity for each color is measured and controlled for, the ratio becomes. This in turn

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