Thyroid divergence in sticklebacks (CBX139). unidentified

Gills were collected from two families of pure marine crosses (Marine Cross 1 and Marine Cross 2) and two families of pure stream-resident crosses (Stream Cross 1 and Stream Cross 2) during both the short and long photoperiods. Gills were stored at -70??C until use. Total RNA was isolated with Trizol Reagent (Invitrogen, Carlsbad, CA, USA), followed by purification with RNeasy Micro Kit (Qiagen, Valencia, CA, USA). Equal amounts of RNA from males and females were pooled for each family: for the short photoperiod samples, two males and two females were pooled for both Marine Cross 1 and Marine Cross 2, four males and four females were pooled for Stream Cross 1, and three males and three females were pooled for Stream Cross 2; for the long photoperiod samples, four males and four females were pooled for Marine Cross 1, one male and one female were pooled for Marine Cross 2, three males and three females for Stream Cross 1, and five males and five females for Stream Cross 2. For each RNA pool, a one-color microarray analysis was performed using a custom-made microarray created by Agilent Technologies (Santa Clara, CA). The microarray contains 43,392 unique oligonucleotide probes representing 19,274 genes (93% of the estimated total gene number of stickleback) [9], as well as additional 37 probes representing nine thyroid- and prolactin-related genes (DIO2, DIO3, IYD, PRLR1, PRLR2, THRA1, THRA2, TSHR1,and TSHR2) designed with eArray software (Agilent Technologies, Santa Clara, CA). The experiments and data normalization were conducted by DNA Chip Research Institute (Yokohama, Kanagawa, Japan). After the RNA quality was checked with a Bioanalyzer (Agilent Technologies, Santa Clara, CA), RNA was labeled with Cy3 and hybridized to the array using a Gene Expression Wash Pack (Agilent Technologies, Santa Clara, CA) and acetonitrile (Sigma, Tokyo, Japan). The arrays were scanned with the Agilent DNA microarray scanner (Agilent Technologies, Santa Clara, CA).