employed in preparing the microarray targets. Antisense amplified RNA was produced from 500 ng of purified total RNA per sample using the figure 2 Amino Allyl MessageAmpTM II aRNA Amplification Kit, as per manufacturers instructions, followed by Cy3 or Cy5 fluor incorporation mediated by a dye coupling reaction, Inhibitors,Modulators,Libraries as previously described in detail. Experimental samples and the pooled reference sample were labelled with Cy3 and Cy5 dye sus pension stocks, respectively. Unincorporated dye was removed by column purification. Dye incorporation and aRNA yield were quantified by spectrophotometry Inhibitors,Modulators,Libraries and further quality controlled by separating 0. 4 uL of the sample through a thin mini agar ose gel and visualising products on a fluorescence scan ner. Microarray hybridisations were performed in a Lucidea semi automated system, without a pre hybridisation step.
For hybridisation of each array, each labelled biological replicate and corresponding pooled reference were com bined and added to the hybridisation solution, compris ing 185 uL 0. 7X UltraHyb buffer, 20 uL poly at l0 mg mL, Inhibitors,Modulators,Libraries 10 uL her ring sperm at c. 10 mg mL and 10 uL ultra pure BSA at 10 mg mL, as detailed previously. Two post hybridisation automatic washes followed by six manual washes to a final stringency of 0. 1�� SSC were performed before scanning. Scanning was performed at 10 um resolution using an Axon GenePix 4200AL Scanner with laser power con stant and auto PMT enabled to adjust PMT for each channel such that less than 0. 1% of features were saturated and that the mean intensity ratio of the Cy3 and Cy5 signals was close to one.
BlueFuse software was then used to identify fea tures and extract fluorescence intensity values from the resultant TIF images. Following a manual spot removal procedure and fusion of duplicate spot data, the resulting fluorescence intensity data and quality annotations Inhibitors,Modulators,Libraries for the 17,102 gene features, were exported into the GeneSpring GX version 10. 0. 2 ana lysis platform after undergoing a block Lowess normalisation. Data transformation and quality filtering were then per formed and all control features were excluded from subse quent analyses. This returned a list of 14,772 genes eligible for statistical analysis. Experimental annotation complied fully with minimum information about a micro array experiment guidelines.
The experi mental hybridisations and Brefeldin_A further methodological details are archived on the EBI ArrayExpress database under accession number E TABM 1089. RT qPCR Expression of selected genes was determined by reverse transcription quantitative real time PCR. Details on the target qPCR primer sequences are given in Table 5. In addition, amplification of three potential refer ence genes fairly cofilin 2, elongation factor 1a and b actin was performed. However, only cofilin 2 expression proved to be sufficiently stable across treatments for nor malisation of the results. Cofilin 2 had been established in a previous salmon cDNA microarray study as a suitable reference gene on