Amplification, labelling and hybridisation of RNA to microarray slides

microarray experiment (MIAME) guidelines (Brazma et al., 2001). Amplified mRNA (aRNA) obtained from normal gill tissue from AGD-naïve fish (AGD- no lesion), gill

lesions from AGD-affected fish (AGD+ lesion) and “normal” gill tissue from AGD-

affected fish (AGD+ no lesion) was hybridised to a single print batch of the salmonid 16K Microarray Version 2.0 slides (von Schalburg et al., 2005). Paired samples were

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labelled with different fluorophores before hybridising to the microarray slides and each biological replicate was reversed (dye flip) to compensate for cyanine fluorophore bias.

Gill tissue from fish sampled at 36 DPE to N. perurans was dissected under a

stereomicroscope in order to isolate either normal tissue or AGD-affected tissue. AGD- affected tissue consisted of gill lesions from AGD-affected fish (AGD+ lesion) while normal tissue from AGD-affected fish (AGD+ no lesion) and AGD-naïve fish (AGD- no

lesion) did not exhibit any evidence of hyperplastic change. Total RNA was purified from each sample using an RNeasy Mini Kit (Qiagen, Doncaster, Victoria, Australia) including an on-column DNase I (Qiagen) DNA digestion step according to the manufacturer’s instructions. Total RNA concentrations were determined using a

spectrophotometer and RNA integrity was verified by agarose gel electrophoresis.

To obtain adequate quantities of RNA for hybridisation experiments, 2 ìg of total RNA

from each fish was subjected to one round of amplification by using the MessageAmp aRNA Amplification Kit (Ambion, Scoresby, Victoria, Australia). The manufacturer’s

instructions were followed throughout except that dUTP was substituted with a mixture of amino allyl-dUTP (Sigma–Aldrich, Castle Hill, NSW, Australia) and dUTP at a ratio

of 1:1 during RNA amplification. Dye coupling to aRNA was performed using either Cy3 or Cy5 according to the Amino Allyl MessageAMP Kit (Ambion) protocol with minor modifications. Briefly, each aRNA sample (3 µg) was dried to completion in a

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(Amersham Biosciences, Castle Hill, NSW, Australia) was added for 30 min in darkness. To quench the reaction, 4 M hydroxylamine (2.25µL, Sigma-Aldrich) was

added followed by the addition of nuclease-free water to a final volume of 30 µL.

Finally, dye-labelled aRNA was filter-purified according to the Amino Allyl MessageAMP Kit (Ambion) instructions.

All microarray slide pre-hybridisation procedures were as previously described (Morrison et al., 2006a). Labelled aRNA was hybridised to pre-warmed microarray slides in a formamide-based buffer (25% formamide, 4× SSC, 0.5% SDS, 2× Denhardt’s

solution and 4 ìL of Genisphere LNA dT blocker) for 16 h at 49°C. Post-hybridisation

washes of the microarray slides were also as previously described (Morrison et al., 2006a). Images of the hybridised microarray slides were acquired immediately at 10 ìm

resolution using a ScanArray Express (PerkinElmer, Fremont, California, USA) slide scanner. The Cy3 and Cy5 fluorophores were excited at 543 and 633 nm respectively and at the same laser power (90%). The photomultiplier tube settings were adjusted between slides to balance the Cy5 and Cy3 channels. Fluorescence intensity data were extracted from TIFF-formatted images using Imagene 5.6.2 software (BioDiscovery, El Segundo, California, USA).

Data analyses were performed in GeneSpring GX (Silicon Genetics, Redwood City, California, USA). For these analyses, background-corrected data were Lowess

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equal to the average base/proportional value in the raw channel were retained. For the purposes of this study, features were incorporated into the data set if they were;

1. Significantly dysregulated in AGD-affected tissue as determined using a Student’s t-test with Benjamini and Hochberg false discovery rate multiple test

correction (MTCBH) (Benjamini and Hochberg, 1995) (p < 0.05), 2. ≥2-fold dysregulated, and

3. Satisfied criterion 1 and 2 in all biological replicates.

The raw data set has been deposited into the GEO (http://www.ncbi.nlm. nih.gov/geo, platform GPL2716 and series GS9595).

The identification (ID) of features on the salmonid 16K Microarray Version 2.0 was initially assigned by the GRASP consortium (http://web.uvic.ca/cbr/grasp). The IDs of all features described in the current study were manually updated using the same process described by the GRASP consortium (von Schalburg et al., 2005). Briefly, Phrap-

assembled contigs for each expressed sequence tag (EST) were updated with data from the ongoing GRASP consortium clustering project (Rise et al., 2004b). The ID of each contig was determined using BLASTX or BLASTN (Altschul et al., 1990) interrogation of non-redundant protein or nucleotide sequences in the GenBank database respectively (http://www.ncbi.nlm.nih.gov/). The IDs were filtered according to the criteria used by the GRASP consortium, so that the lowest-scoring significant hit (E < 10-15) in BLASTX was chosen to represent the EST; otherwise the lowest-scoring BLASTN hit was chosen to represent the EST.

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6.3.3 RNA extraction and cDNA synthesis for quantitative real-time PCR (qRT- PCR)

For microarray data verification, 800 ng of AGD+ lesion and AGD- no lesion aRNA from the microarray experiment was reverse transcribed using Superscript III reverse transcriptase and random hexamers (Invitrogen) according to the manufacturer’s

protocol. The aRNA cDNA template was diluted 80-fold to 1600 ìL for further studies.

Two undiluted samples of cDNA from AGD+ lesion and AGD- no lesion aRNA were pooled in equal proportions and used as cDNA template to calculate the PCR

amplification efficiency of each gene of interest.

Total RNA was isolated from AGD+ lesion, AGD+ no lesion and AGD- no lesion RNAlater-stabilised gill tissues sampled at 12, 25 and 36 DPE to N. perurans. Total RNA was purified using an RNeasy Mini Kit (Qiagen), a Dounce homogeniser

(Wheaton Scientific, Millville, NJ) and QIAshredders (Qiagen). All RNA was DNase I- treated using Turbo DNA-free (Ambion). RNA extraction and DNase I treatment were performed according to the manufacturer’s instructions. The quality and quantity of total

RNA was measured as described above. To determine the relative expression of genes at various times post-exposure to N. perurans, total RNA was reversed transcribed from samples obtained at 12 (120 ng), 25 (800 ng) and 36 (800 ng) DPE as described above except random hexamers were replaced by a mix of ten parts random hexamers (Invitrogen) (50 ng) and one part oligo(dT)20 (Invitrogen) (1 ìL of 50 ìM stock) as

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advised by Quantace (Neutral Bay, New South Wales, Australia). cDNAs were diluted 3-fold (12 DPE) or 8-fold (25 and 36 DPE) for further studies. Three undiluted samples of cDNA from AGD+ lesion and AGD- no lesion 36 DPE to N. perurans were pooled in equal proportions and used as cDNA template to calculate the amplification efficiency of each gene of interest.