source: wild population developmental stage: stage 10 embryo
Treatment protocol
Pools of frozen embryos collected at each developmental stage were used for RNA isolation, labeling, and microarray hybridization. Four pools of 25 embryos were used for stages 1-10, four pools of 15 embryos were used for stages 11-15, and 4 pools of 10 embryos were used for stages 16-40.
Growth protocol
Eggs from multiple females were fertilized by sperm from multiple males, and excess sperm were removed. Fertilized embryos were maintained in Petri dishes half submerged in 15 ppt filtered seawater in a 25°C environmental chamber under light during the first two stages of development (2-cell stage). Embryos that successfully reached the 2-cell stage within 3 hours were incubated under a 16 hour light: 8 hour dark photoperiod at 25°C in the environmental chamber (818 Low Temperature Illuminated Incubator, Precision Scientific, USA).
Extracted molecule
total RNA
Extraction protocol
Embryo RNA was extracted using a Trizol buffer (Invitrogen, Carlsbad, CA, USA) followed by purification using the Qiagen RNeasy Mini Kit (Qiagen Inc., Valencia, CA, USA).
Label
Cy3
Label protocol
RNA for hybridization was prepared by one round of amplification (aRNA) using the Amino Allyl MessageAmp aRNA Kit (Ambion, Austin, TX, USA) to form copy template RNA by T7 amplification. Amino-allyl UTP was incorporated into targets during T7 transcription, and resulting amino-allyl aRNA was coupled to Cy3 and Cy5 dyes (GE Healthcare, Piscataway, NJ, USA).
Hybridization protocol
Labeled aRNA samples (2 pmol dye/ul) were hybridized to slides in 10 ul of hybridization buffer [50% formamide buffer, 5x SSPE, 1% sodium dodecyl sulfate, 0.2 mg/ml bovine serum albumin, 1 mg/ml denatured salmon sperm DNA (Sigma), and 1 mg/ml RNAse free poly(A) RNA (Sigma)] for 44 hours at 42º C.
Scan protocol
Arrays were scanned using a ScanArray Express 4000 (Perkin Elmer). Resulting 16 bit Tiff Images were quantified using ImaGene® (Biodiscovery, Inc.) spotfinding software.
Data processing
Log2 measures of gene expression were normalized using a linear mixed model in JMP Genomics 3.2 (SAS, Cary, NC, USA) to remove the effects of dye (fixed effect) and array (random effect) following a joint regional and spatial Lowess transformation in MAANOVA version 0.98.8 for R to account for both intensity and spatial bias. The model was of the form yij = μ + Ai + Dj + (AxD)ij + εij where, yij is the signal from the ith array with dye j, μ is the sample mean, Ai and Dj are the overall variation in arrays (arrays 1-80) and dyes (Cy3 and Cy5), (AxD)ij is the array x dye interaction and εij is the stochastic error.