RNA samples Multiple myeloma were pooled across subjects in order to reduce the effect of biological variation. A formula, that dictates the total number of subjects and arrays required for the pooled experiment to obtain gene expression estimates and confidence intervals comparable to those obtained from a non pooled experiment, gave 90% confi dence if nine subjects were pooled across a total of three arrays. To this effect, equal amounts of total RNA from three crabs in one moult stage, were pooled, and compared against equal amounts of total RNA pooled from three crabs in another moult stage, on one array. This was repeated three times in total, the different moult stages were labelled with Cy3 or Cy5 respectively.

Consecutive moult stages were compared in the follow ing format, post moult with intermoult, intermoult with early pre moult, early pre moult with late pre moult, late pre moult with ecdysis, and ecdysis with post moult. Figure 2 is a schematic diagram depicting each set of moult stage comparisons. Spatial variation within each array was addressed through spot duplication. Two identical blocks of grids consisting of each amplified cDNA and including the controls described above were printed onto the left and right sides of each horizontally orientated array, thus affording spatial separation between duplicate spots, to allow for the normalisation of potential hybridisation anomalies. Nine small crabs were snap frozen, individually ground under liquid nitrogen and RNA was isolated from each ground crab using TRIZOL reagent as recommended by the manu facturer.

The RNA was DNase treated using RQ1 RNase free Dacomitinib DNase according to the manufacturers instructions and puri fied using RNeasy Mini Kit as recommended by the manufacturer. RNA quality was assessed by visualisation on a denaturing formaldehyde RNA gel using ethidium bro mide staining. Concentration and purity of the RNA were determined by measuring the absorbance at 260 nm and 280 nm using a spectrophotometer. One microgram of Lucidea universal RNA control was added to 10 ug of pooled total RNA for each moult stage sample, the RNA was con verted to cDNA then labelled and hybridised to the array using the 3DNA Array 900 MPX expression array detection kit according to the manufacturers protocol. Briefly, RNA was reverse transcribed using a random primer com bined with an oligo dT primer. The RNA was then degraded and the cDNA tailed with dTTP followed by ligation to a dendrimer specific capture oligo. Microarray slides were denatured prior to use by immersion in 95 C MilliQ water for 5 min, the slides were then transferred to 95% ethanol at room temperature for 2 min. Slides were spun dry to reduce streaking at 800 RPM for 2 min.