Project description:Early life stage mortality is an important issue for Atlantic cod aquaculture. The impact of the cod maternal (egg) transcriptome on egg quality and mortality during embryonic development is poorly understood. In the present work we studied embryonic mortality and maternal transcript expression using 15 females within an Atlantic cod broodstock development program. Cumulative mortality at 7 days post-fertilization (segmentation stage) and percent hatch were used to assess embryonic survival as an indicator of egg quality. A 20,000 probe (20K) microarray experiment compared the 7 hour post-fertilization (7 hpf, ~ 2-cell stage) egg transcriptome of the two lowest quality females (both with less than 1 percent hatch) to that of the highest quality female (greater than 50 percent hatch). Overall design: At 7 hours post-fertilization, 3 batches of 0.25 ml eggs were collected from each of the two lowest quality females (females 12 and 13) and the highest quality female (female 2). For each female, total RNA was isolated from each of the three batches separately, and pooled before microarray analysis. Two 4-array direct comparison experiments were performed. For each 4-array experiment, one of the ‘low quality’ females was compared to the ‘high quality’ female, and each RNA was labeled in 4 labeling reactions: two with AlexaFluor 647 and two with AlexaFluor 555. Each 4-array experiment consisted of four technical replicates: two duplicates and two dye-swaps. Females 12 and 13 are two biological replicates.
Project description:The Atlantic cod, Gadus morhua, is an important species both for traditional fishery and increasingly also in fish farming. The Atlantic cod is also under potential threat from various environmental changes such as pollution and climate change, but the biological impact of such changes are not well known, in particular when it comes to sublethal effects that can be difficult to assert. Modern molecular and genomic approaches have revolutionized biological research during the last decade, and offer new avenues to study biological functions and e.g. the impact of anthropogenic activities at different life-stages for a given organism. In order to develop genomic data and genomic tools for Atlantic cod we conducted a program were we constructed 20 cDNA libraries, and produced and analyzed 44006 expressed sequence tags (ESTs) from these. Several tissues are represented in the multiple cDNA libraries, that differ in either sexual maturation or immulogical stimulation. This approach allowed us to identify genes that are expressed in particular tissues, life-stages or in response to specific stimuli, and also gives us information about potential functions of the transcripts. The ESTs were used to construct a 16 k cDNA microarray to further investigate the cod transcriptome. Microarray analyses were preformed on pylorus, pituitary gland, spleen and testis of sexually maturing male cod. The four different tissues displayed tissue specific transcriptomes demonstrating that the cDNA array is working as expected and will prove to be a powerful tool in further experiments.
Project description:This study was performed to validate the newly developed CGP Atlantic cod 20K oligonucleotide microarray. Atlantic cod (Gadus morhua) received an intraperitoneal injection of either formalin-killed, atypical Aeromonas salmonicida (Asal) or PBS and transcriptional profiles of spleen tissues from Asal-injected fish were compared to those from pre-injection control fish and PBS-injected control fish. Gene expression profiles resulting from this study were compared to those from suppression subtractive hybridization library studies, that were previously performed on the same samples, and to literature. Gene expression patterns of single genes were confirmed by QPCR analysis. This study has shown that the newly developed CGP Atlantic cod 20K oligo microarray platform is a valuable tool for cod genomic research. Overall design: Fish were assigned to 1 of 3 groups: Asal group, PBS group or 'undisturbed control' group (the latter were not handled during the experiment). These groups were kept in 3 separate tanks. For the test samples, RNA was used from individual spleen samples that were taken from 6 fish from each of 4 treatment groups: PBS group pre-injection (0H PBS), Asal group pre-injection (0H Asal), PBS group 24 hours post-injection (24HPI PBS) and Asal group 24 hours post-injection (24HPI Asal). All test samples were labeled with AlexaFluor 647. For the universal reference sample, RNA from 31 'undisturbed control' fish was pooled, with each individual contributing an equal amount, and labeled with AlexaFluor 555. Each individual test sample was hybridized together with the universal reference sample on an array. For one sample from the 0H PBS group the array failed. This study therefore includes 6 biological replicates for 0H Asal, 24HPI Asal and 24HPI PBS groups and 5 biological replicates for the 0H PBS group.