gender: female development stage: spawning tissue: muscle tissue source: beach
Extracted molecule
total RNA
Extraction protocol
The samples were thawed and blotted with a Kimwipe®. All of the primary locomotion muscle tissue including red and white muscle tissue was removed from each fish. Total RNA was extracted with a modified protocol of the Invitrogen TRIzol ® Plus RNA purification kit using PureLinkTM Micro-to-MidiTM columns. Disruption and homogenization were achieved with a MixerMill MM301 (Retsch). The manufacturer's protocol was followed for each extraction with the exception of using 150 ul of chloroform and 150 ul of low pH phenol to ensure dissociation of proteins and isolation of RNA. The quality of all RNA samples was verified on a 1% agarose gel. All samples were quantified with a Spectrophotometer ND-1000 (NanoDrop).
Label
Cy5
Label protocol
Indirect cDNA labeling system kits per manufacturer's instructions. In brief, 10 ug of total RNA from a single individual was combined with a master mix includingreverse-transcriptase and oligo (dT) 20 primers. This reaction was incubated for three hours at 46°C tosynthesize single-stranded cDNA. The samples were cleaned with the S.N.A.P TM column purification procedure (Invitrogen). A reference pool was prepared with representative total RNA samples of juvenile sockeye muscle and liver, and adult sockeye brain, muscle and liver. Though this reference pool was hybridized to the arrays, this chanel (Cy3) was ignored fro this experiment and the signal (Cy5) channel was treated as a single color experiment. Individual samples and were coupledwith mono-reactive CyDyeTM packs (GE Healthcare). In short, the common reference pool of aRNA was coupled with Cy3 and the individual sockeye muscle tissue cDNA with Cy5 dyes for one hour at 4°C. The samples were then purified to remove all uncoupled dye using S.N.A.P.TM columns as per manufacturer's instructions (Invitrogen). The dye coupled sample and reference were stored at 4°C in the dark until hybridization.
Hybridization protocol
We used the cGRASP 16 k cDNA microarray to compare the transcriptomes of these populations with divergent life histories. This microarray consists of 16,006 elements chosen from 300,000 Atlantic salmon and rainbow trout cDNA libraries. The libraries were derived from a variety of tissue types at different development stages, and conditions. Element sequences were chosen for minimum overlap, sequence quality, andother criteria. We followed an established hybridization protocol for the 16 k cDNA array [50]. In brief, both 250 ng of reference aRNA and 500 ng of sample cDNA were collectedin a single tube and kept dark. The mixture was concentrated with a speed vacuum and brought up to 23 ul with RNase free water (Gibco). Hybridization buffer #3 (Ambion) was heated to 65°C while occasionally mixing for one hour. The heated buffer and LNAdt blocker (Genesphere) was then added to the collected sample, as per manufacturer's instructions. We used the Tecan HS 4800 Pro, an automated hybridization machine to hybridize sample cDNA to the arrays (Tecan). Before the sample injection, the programmed Tecan washed with several solutions containing first 1 × SSC, then 0.1 × SSC 0.014% SDS, then 5 × SSC, 0.01% SDS, and 0.2% BSA. Samples were heated to 80°C for 5 minutes, and then kept at 65°C until injected onto the pre-washed arrays in a Tecan HS 4800 Pro, as per manufacturer's instructions. Microarrays were hybridized for 16 hours. Post-hybridization, arrays were rinsed in the Tecanmodules with increasingly stringent SSC and SDS solutions, starting with 2 × SSC, 0.014%SDS for four washes incrementally decreasing temperature, then one final wash of 0.2 × SSC at 23 °C. Finally, slides were dried with 37 psi nitrogen gas and kept dark until scanned.
Scan protocol
All microarrays were scanned immediately after hybridization was complete using a ScanArray Express (Perkin-Elmer). The microarray images were quantified manually with ImaGene 5.6.1 (BioDiscovery). Spots with unusual morphology, offset, or other poor quality parameters were flagged as marginal and excluded these from downstream analyses.
Description
SAMPLE 8
Data processing
Only the signal Cy5 channel was considered for this experiment. The reference Cy3 channel was ignored. Normalization was performed using the Limma R package BetweenArrayNormalization function with the Quantile method
