Lr J1

Fig. 4.4. Feature density options of NimbleGen arrays. A variety of feature sizes and densities are available on NimbleGen arrays. In this experiment, identical probes specific for a subset of mouse genes were arrayed in four different probe formats on a single array and hybridized with cRNA derived from mouse liver total RNA.

Tab. 4.1 Array designs available with the NimbleGen gene expression platform.

Array format

Total probes

Feature size

Pitch

Illustration

1_2

393 216

16 ^m

34 ^m11

Figure 4.4 C

1_4

196 608

16 ^m

34 ^m

Figure 4.4 B

4_4

196 608

33 ^m

34 ^m

Figure 4.4 D

4_9

87 296

33 ^m

51 ^m

Figure 4.4 A

1) 24 ^m centre-to-centre spacing on the diagonal.

1) 24 ^m centre-to-centre spacing on the diagonal.

active mirrors, resulting in four active mirrors in a total group of nine used to synthesize a single spot; thus, this format is referred to as 4_9. By varying the number of grouped mirrors and the proportion of dormant mirrors used as spacers, mi-croarrays with a large number of possible spot sizes and pitches can be created, such as the 1_4, 1_2, and 4_4 formats shown in Figure 4.4 B-D, respectively.

Table 4.1 details the standard array formats offered by NimbleGen. As many as 393 216 features (probes) can be synthesized on a single custom array in the highest density 1_2 format. In any of these formats, the oligonucleotides can vary in length from 1-mers up to a maximum of 80-mers. Since all spots are synthesized in parallel, spot size, pitch, and feature quantity do not affect manufacturing costs.

On a typical array, probes are placed within a defined layout of control elements. These control elements are designed to control for possible variations in the synthesis and/or hybridization of the array. Figure 4.5 demonstrates the layout and number of these control probes. As shown in the figure, the 'A' probes are used to test overall uniformity of the array synthesis, and the corner probes also serve as fiducial marks for automated image analysis (image location, grid alignment, and feature intensity extraction). More than 600 'A' probe replicates are distributed throughout the entire array. 'B' probes are base-specific synthesis controls, represented 112 times on the array. 'C' probes are a six-point spike-in standard curve that demonstrates the limit of detection (sensitivity) and linear range of the array. These sets are replicated 56 times throughout the array. 'D' probes are a 10-point photometric standard curve used to assess synthesis efficiency with varying light dosage and are replicated 12 times on the array. In total more than 1200 quality-control probes are incorporated into every array.

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