For all microarray experiments, cDNAs used for hybridisation were prepared from 20 µg samples of total RNA from a single tissue type (gill, intestine, brain and renal kidney) either pooled from all animals in an experimental group (n=6) or from individual fish. Two cDNA samples from different eel groups were co-hybridised to a microarray, one sample being labelled with Cy3 and the other with Cy5. Experiments were replicated in dye- swap to address any labelling bias caused by differential incorporation rates between Cy3 and Cy5 (Churchill, 2002). Dye-swap replication involves repetition of the entire microarray experiment but swapping the cDNA labels so that the cDNA previously labelled with Cy3 is now labelled with Cy5 and vice versa. This controls for any bias which could be introduced if the Cy3 and Cy5 labelling reactions are not equally efficient.
Fluorescently labelled cDNA was created using the CyScribe Post- Labelling Kit (Amersham Biosciences, Little Chalfont, UK) according to the manufacturers instructions. CyScript™ reverse transcriptase is used to create first-strand cDNA incorporating a chemically reactive nucleotide analogue (amino allyl-dUTP) into the cDNA. The RNA is then degraded and the cDNA purified to remove free nucleotides and then labelled with the reactive forms of Cy3 or Cy5 NHS-esters that bind to the modified amino allyl-nucleotides. After a final purification, the labelled cDNA is ready for hybridisation.
cDNA production and labelling: Briefly, two first strand synthesis reactions were performed in parallel for the two samples to be hybridised to the microarray. Eel RNA (20 µg) was combined with random nonamer primers (1 µl, Amersham Biosciences) and/ or anchored oligo d(T) primer (1 µl) and the volume made up to 11 µl with H2O. Both primer types were used for amplified mRNA but only the anchored oligo d(T) was used with total RNA to avoid non-messenger RNA being transcribed. Reactions were incubated at 70 °C for 5 min and then cooled and left at room temperature for 10 min to
mix (1 µl, proprietary), CyScribe™ post -labelling amino allyl-dUTP (1 µl, proprietary) and CyScript™ reverse transcriptase (1 µl). The reaction was incubated at 42 °C for 90 min to allow reverse transcription to occur before cooling to 37 °C and adding NaOH (2 µl, 2.5 M). The reaction was incubated for 15 min to degrade the RNA and then neutralised by adding 10 µl HEPES (2 M, pH 6.6). The cDNA was purified by adding to a GFX glass fibre matrix purification column (Amersham Biosciences) containing 500 µl GFX capture buffer (proprietary) and centrifuging over a 1.5 ml micro-centrifuge tube at 13800 g for 30 seconds. The eluate was discarded and the column transferred to a fresh 1.5 ml tube. The cDNA was washed three times by adding 600 µl ethanol (80 %) and centrifuging at 13800 g for 30 seconds, discarding the eluate after each centrifugation. The column was centrifuged for an additional 10 s at 13800 g to remove residual ethanol and then
transferred to a fresh 1.5 ml tube. To elute the cDNA, freshly made sodium bicarbonate (60 µl, 0.1 M, pH 9.0) was added to the column and incubated at room temperature for 5 min. The column was centrifuged again at 13800 g for 1 min to collect the purified amino allyl-labelled cDNA. The cDNA was post-labelled by using the amino allyl-labelled cDNA solution to resuspend an aliquot of Cy3 or Cy5 CyDye NHS-ester (Amersham Biosciences) and
incubated in the dark at room temperature for 90 min. Unreacted CyDye NHS-ester was inactivated by adding hydroxylamine (15 µl, 4 M) and incubated in the dark at room temperature for a further 15 min.
Unincorporated CyDye was immediately removed from the fluorescently- labelled cDNA by using a CyScribe GFX purification column as described above except that GFX wash buffer (Amersham Biosciences) was used instead of 80 % ethanol to wash the cDNA.
The two differentially labelled (Cy3 and Cy5) cDNA samples were mixed in a light-proof 1.5 ml tube, to which was added calf thymus DNA (20 µl, 200 ng/ ml) and poly A oligo-nucleotides (2 mg/ml, 20-mers). The solution was transferred to a Centricon concentrator column (Millipore, Watford, UK) supported in a 1.5 ml micro-centrifuge tube. Water was added up to the mark engraved on the column and then centrifuged at 14000 g for 5 min. The eluate was discarded and H2O again added to the engraved mark and the column centrifuged at 14000 g for 8 min. Further centrifugations at 14000 g
were performed for 1 min until the remaining volume was 14.4 µl or less. The solution was collected by placing the column inverted in a fresh 1.5 ml micro- centrifuge tube and centrifuging for 1 min at 14000 g. The solution was transferred to a PCR tube, combined with 20 x SSC buffer (15 µl), formamide (30 µl), SDS (0.6 µl, 10% weight:volume) and the volume made up to 60 µl with H2O. The solution was heated to 95 °C for 5 min to denature the DNA and then snap cooled on ice before being applied to a pre-hybridised microarray slide as described below.
Microarray pre-hybridisation: Microarrays were pre-hybridised for 1 hour at 45 °C in 50 ml pre-hybridisation solution containing to following final concentrations; formamide (50 % v/v); 5 x SSC buffer; SDS (0.1 %); bovine serum albumin (0.1 mg/ ml); denatured calf thymus DNA (200 ng/ ml) and yeast RNA (200 ng/ ml). Pre-hybridisation reduces background fluorescence signal which usually originates from non-specific hybridisation of the labeled samples or auto-fluorescence of the glass slide. Arrays were washed twice for 5 min in 0.1 x SSC for 5 min, dipped in H2O and dried by centrifugation. Labelled cDNA probes were aliquoted onto the arrays and overlayed with an M-series lifterslip (Erie Scientific Company, Portsmouth, USA) which ensures equal distribution of probe across the printed surface as they have a raised perimeter edge on the underside. Arrays were placed in an airtight chamber to maintain humidity and incubated overnight at 44 °C to allow binding of the labelled cDNA with the complementary features on the microarray.
Microarray stringency washes: The microarray was placed vertically into a flask containing solution A (50 ml, 10 % SDS, 1 x SSC) at room
temperature to allow the coverslip to come away from the microarray. The microarray was transferred to a cylindrical flask containing solution A (50 ml) at room temperature and placed on a roller for 5 min. The microarray was then transferred to a fresh flask containing solution B (50 ml, 1 % SDS, 1 x SSC) pre-warmed to 42 °C and placed on a roller inside an incubation oven at
with solution D (50 ml, 0.01 x SSC) at room temperature by submerging for 10 s. The array was dried quickly by placing in a slide rack and centrifuging at 2000 g for 1 min and scanned immediately.