Fig. 4. Assembly of the microarray chip and multigene expression patterns related to elevation of ecdysteroids by two methods of endocrine molt induction. (A) Schematic representation of the hybridization design of the experiment showing each of the biological replicates. Arrowhead represents Cy3 labeling, and tail represents Cy5 labeling. XO–SG, premolt crayfish induced by XO–SG removal; 20E, premolt crayfish induced by repetitive 20E injections. The different colors of the circles in each treatment group represent different individuals, whereas in the reference group the same color represents pooling of the samples. (B) Overview of all the clustered genes according to treatments in the experiment. Red, treatment higher than reference; green, reference higher than treatment; yellow, equal expression. Two representative clusters of the ecdysteroid-responsive genes are indicated by boxes: the green box marks the downregulated cluster and the red box the upregulated cluster. (C) Expression scatter plots of all the genes represented on the chip in the two treatments. M, log₂-fold change of normalized emission intensity between the treatment and the control; A, average signal in all treatments; green line, the minimum threshold of A at 8.5; red spots, genes with P<0.05, M value >1 or <–1, and A value >8.5.