The study of Atlantic cod (Gadus morhua) spleen global gene expression response to intraperitoneal injection with formalin-killed atypical Aeromonas salmonicida.

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. 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.

本研究旨在验证新开发的CGP大西洋鳕20K寡核苷酸微阵列（CGP Atlantic cod 20K oligonucleotide microarray）。实验选取大西洋鳕（Gadus morhua），对其腹腔注射甲醛灭活的杀鲑气单胞菌非典型株（Asal）或磷酸盐缓冲液（PBS），并将注射Asal的鱼的脾脏组织转录表达谱与注射前对照鱼及PBS注射对照鱼的转录表达谱进行比对。本研究得到的基因表达谱，与此前基于相同样本开展的抑制性消减杂交文库（suppression subtractive hybridization library）研究结果及公开文献数据进行了比对。单基因的基因表达模式通过定量聚合酶链反应（QPCR）分析得到验证。本研究证实，新开发的CGP大西洋鳕20K寡核苷酸微阵列平台是用于鳕基因组研究的可靠工具。受试鱼被分为3组：Asal注射组、PBS注射组及未干预对照组（该组实验全程未接受任何操作），3组分别饲养于3个独立水族箱中。本次检测样本取自4个处理组的个体脾脏组织RNA，每个处理组分别为：PBS注射前组（0H PBS）、Asal注射前组（0H Asal）、PBS注射后24小时组（24HPI PBS）及Asal注射后24小时组（24HPI Asal），每个组取6尾鱼的样本。所有检测样本均采用AlexaFluor 647进行荧光标记。通用参考样本则由31尾未干预对照鱼的脾脏RNA等量混合制备，每份个体RNA贡献等量体积，随后采用AlexaFluor 555进行荧光标记。将每份检测样本与通用参考样本混合后，在微阵列芯片上完成杂交反应。其中1份0H PBS组的样本杂交失败，因此本研究的生物学重复设置为：0H Asal、24HPI Asal及24HPI PBS组各6个生物学重复，0H PBS组为5个生物学重复。