Multiplex Array Consistency

One requirement for prospective toxicogenomics screening is that the array results observed for the training set be consistent with those observed for the screening set. In addition, any array-based screening method required that arrays demonstrate a high degree of intra- and inter-array signal consistency. Although global shifts in intensity can, to a certain extent, be adjusted by dataset normalization, the best array results are obtained from the most consistently manufactured arrays.

The reproducibility of 12-well hybridizations has been tested by comparison of inter-well correlations of replicate hybridizations. Table 4.2 is an example comparison of mouse liver cRNA hybridized to a set of genes selected from mouse liver and spleen. The average difference values for every gene in the set were compared between each well and the remainder of the wells in the array by a least-squares fit linear correlation. As can be seen in the table, the data were highly reproducibly from well to well with an average R2 value of 0.997. The same level of reproducibility is achieved when comparing 1-plex and 12-plex data for comparable probe sets. The fact that the results are highly consistent in the transition from the full array to the 12-well format provides good assurance that the results observed during training set development will hold during routine screening set deployment.

Tabl. 4.2 Well-to-well dataset reproducibility. Twelve replicates of a mouse liver probe set containing 6789 features were synthesized in a 12-plex array and hybridized with 12 aliquots from the same sample. The data were extracted and average difference values for each gene were calculated. The datasets for each well were compared by least-squares fit linear correlation, and the inter-well R2 values are shown in the table. The average R2 value for the set was 0.9972.

Well 1

Well 2

Well 3

Well 4

Well 5

Well 6

Well 7

Well 8

Well 9

Well 10 Well 11

Well 12

Well 1

1.000

0.998

0.997

0.997

0.997

0.996

0.996

0.998

0.996

0.995

0.994

0.996

Well 2

-

1.000

0.998

0.998

0.998

0.998

0.998

0.999

0.998

0.996

0.996

0.998

Well 3

-

-

1.000

0.997

0.998

0.999

0.997

0.998

0.998

0.996

0.997

0.998

Well 4

-

-

-

1.000

0.999

0.997

0.998

0.998

0.997

0.996

0.997

0.997

Well 5

-

-

-

-

1.000

0.998

0.998

0.998

0.998

0.996

0.998

0.997

Well 6

-

-

-

-

-

1.000

0.998

0.998

0.999

0.997

0.998

0.997

Well 7

-

-

-

-

-

-

1.000

0.999

0.998

0.997

0.998

0.997

Well 8

-

-

-

-

-

-

-

1.000

0.999

0.997

0.997

0.997

Well 9

-

-

-

-

-

-

-

-

1.000

0.996

0.998

0.996

Well 10

-

-

-

-

-

-

-

-

-

1.000

0.998

0.997

Well 11

-

-

-

-

-

-

-

-

-

-

1.000

0.997

Well 12

-

-

-

-

-

-

-

-

-

-

-

1.000

0 0

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