SOURCE POP: Heron Island CHIP #: 18 HYB DATE: 18/4/2006
Growth protocol
Spawning of adult Haliotis asinina and larval culture were carried out at Heron Island Research Station on the Great Barrier Reef, Australia (23°27’S; 151°55’E) as previously described (Counihan et al. 2001; Jackson et al. 2005). Larval cultures were derived from gametic contributions from at least 3 females and 3 males, and were treated for 2 hours daily with 20 μg/L rifampicin to protect against bacterial invasion. Larvae were allowed to develop to 96 hours post fertilisation (hpf), at which time a subset were introduced into 0.5 L plastic chambers containing ~11.5 cm2 surface area of the encrusting coralline algae, Mastophora pacifica, previously shown to be an effective inducer of Haliotis asinina metamorphosis (Jackson et al. 2005). After 2 h, chambers were supplied with a gentle flow of seawater, such that any larvae not in constant contact with the coralline algae were washed out of the chamber. At 8 h after their introduction, the algal shards were removed from chambers and all attached larvae were washed into fresh 0.5 L plastic chambers using a jet of 0.22 μM filtered seawater (FSW). Once again, chambers were supplied with a gentle flow of seawater so that only larvae exhibiting constant crawling behaviour were maintained. Washed larvae were allowed to develop for a further 48 h, at which point the experiment ended. Throughout the induction experiment, postlarvae were treated with rifampicin as described above. A subset of larvae were maintained in the original culture chambers to act as an non-induced control.
Extracted molecule
total RNA
Extraction protocol
Abalone larvae were collected for RNA extraction at 66, 78, 90, 108, 120 and 144 hpf. Competent larvae were induced by exposure to coralline algae at 96 hpf (see above) and postlarval samples were collected at 12, 24 and 48 hpi. This meant that postlarvae 12, 24 and 48 hpi were actually the same age as larvae 108, 120 and 144 hpf. Total RNA was extracted using TRIreagent (Sigma) as described by the manufacturer. Quantity and quality of RNA was assessed using UV spectrophotometry (Nanodrop ND-1000) and agarose gel electrophoresis.
Label
Cy5
Label protocol
2.5 μg total RNA was used to prepare labelled cDNA for each developmental stage. cDNA was transcribed with an oligo(dT)15 primer using a dNTP mix with a ratio of 4:1 aminoallyl-dUTP to dTTP (Sigma) and Superscript III Reverse Transcriptase (Invitrogen) according to manufacturers’ instructions. Aminoallyl-labelled cDNA (aa-cDNA) was purified, dried and redissolved in 100 mM sodium carbonate pH 9. Cy3 and Cy5 reactive dyes (Amersham) were added to their respective aa-cDNA samples and the coupling reaction allowed to proceed. After 1 h, 4.5 μl 4 M hydroxylamine was added to remove unincorporated dyes. Cy3- and Cy5-labeled cDNA were combined and purified with a PCR cleanup kit (Qiagen). To prevent non-specific binding, 10 μg human Cot1 DNA (Invitrogen) and 0.125 A260 units poly dA (Roche) were added to the purified probe mix. This solution was dehydrated in a speed-vac to a volume of 8 μl.
Hybridization protocol
4X SSC, 40% deionized formamide (Astral Scientific) and 0.1% SDS were added to the probe mix to create the hybridisation buffer. Following incubation at 95ºC for 5 min and 45ºC for 90 min, the labelled probe mix was loaded onto the microarray chip, and placed in a hybridisation chamber (Corning) with 10 μl H2O at each end in a 45 ºC oven for 16 h (as per Woods et al. 2004).
Scan protocol
Following hybridisation, chips were washed once in a 0.2X SSC, 0.05% SDS solution and twice in 0.2X SSC solution, then dried by low speed centrifugation. Washed slides were scanned using a GenePix 4000B Microarray Scanner (Molecular Devices). Spot intensities from raw scan data were quantified using GenePix Pro 6.0 software (Molecular Devices). Spots were flagged as present or absent based on circularity and diameter (circularity </= 70 and diameter < 100 = absent) or sum of means (Cy5 intensity/Cy3 intensity < 200 = absent).
Description
loop design
Data processing
Quantified data was transferred as a one-colour analysis into Genespring version 7.0 (Silicon Genetics). Raw data was normalised per chip to the 50th percentile and per gene to the median, and measurements < 0.01 were set to 0.01. Median polishing was performed per chip and per gene.
