microarray summer mortality insitu oyster

Summer mortality of the Pacific oyster Crassostrea gigas is the result of a complex interaction between oysters, their environment and pathogens. Heredity appears to be a major factor determining the sensitivity of oysters to summer mortality, allowing resistant (R) and susceptible (S) lines to be produced. We conducted genome-wide expression profiling of R and S gonads during the 3-month period preceding a summer mortality event using a 9K cDNA microarray that we designed. This transcriptional analysis provides new indications to define markers for Quantitative Trait Loci searches and functional studies, and evaluates the potential role of each gene in the resistance to summer mortality For microarray analysis, R and S oysters were sampled four times (dates 1 to 4: May 9, May 25, June 6, and June 20, respectively). On each date, 3 replicates of 8 oysters were sampled from each line (R and S) and the gonads prepared for total RNA extraction. Furthermore, the entire tissues of 10 wild oysters were collected, pooled and homogenized to constitute a single total RNA sample for use as a reference in all slide hybridizations and RT-PCR analysis. For microarray hybridizations, 5µg of total RNA were directly labeled by reverse transcription and then purified using the Direct ShipShot Labeling kit (Promega). This reaction was performed for each of the 24 gonad samples, with Cy5 (red) incorporation. The reference sample was Cy3-labeled (green) in 24 separate tubes following the same protocol. The 24 Cy3-labeled cDNAs were next pooled, and then divided once more into 24 samples to obtain a homogeneous reference. Equimolar amounts of cDNA samples and cDNA reference labeled with Cy5 and Cy3, respectively, were SpeedVac evaporated and mixed into a single pool with the hybridization buffer (ChipHyb™ hybridization buffer, Ventana Discovery, Tucson, AZ, USA). They were then co-hybridized on the same microarray slide, in a Ventana hybridization station (Ventana Discovery, Tucson, AZ, USA). The data submitted here correspond to the mean of the three replicates for each line and each date, representing 8 samples : gonad_resistant_date1 gonad_sensitive_date1 gonad_resistant_date2 gonad_sensitive_date2 gonad_resistant_date3 gonad_sensitive_date3 gonad_resistant_date4 gonad_sensitive_date4

太平洋牡蛎（Pacific oyster，*Crassostrea gigas*）的夏季死亡现象，是牡蛎、其生存环境与病原体三者间复杂相互作用的产物。遗传因素是决定牡蛎对夏季死亡敏感性的关键因素，借此可培育出抗逆（R）与易感（S）品系。本研究利用自主设计的9K cDNA微阵列（cDNA microarray），在夏季死亡事件暴发前的3个月周期内，对抗逆与易感品系牡蛎的性腺组织开展全基因组表达谱分析。该转录组分析为定量性状位点（Quantitative Trait Loci，QTL）的搜寻与功能研究提供了全新的标记开发思路，并评估了各基因在抗夏季死亡能力中的潜在作用。针对微阵列分析实验，研究人员于4个时间点（日期1至4依次为5月9日、5月25日、6月6日与6月20日）采集抗逆与易感品系的牡蛎样本。每个时间点下，从每个品系中选取8只牡蛎作为3个生物学重复，制备其性腺组织用于总RNA提取。此外，研究人员还收集了10只野生牡蛎的全部组织，经混合均质后制备得到一份总RNA混合样本，作为所有芯片杂交与逆转录PCR（Reverse Transcription PCR，RT-PCR）分析的参照样本。在微阵列杂交实验环节，取5μg总RNA通过逆转录反应直接标记，随后使用Direct ShipShot标记试剂盒（Promega公司）完成纯化。本实验针对全部24个性腺样本均开展了上述标记纯化流程，并以Cy5（红色）进行荧光标记。参照样本则采用相同实验方案，在24个独立反应管中以Cy3（绿色）完成标记。随后将24份Cy3标记的cDNA混合均匀，并再次均分为24份样本，以获得均质化的统一参照样本。将分别以Cy5和Cy3标记的待测cDNA样本与参照cDNA样本按等摩尔浓度混合，经SpeedVac真空浓缩后，与杂交缓冲液（ChipHyb™杂交缓冲液，Ventana Discovery，美国亚利桑那州图森市）混合为单一杂交体系。随后将该混合体系置于Ventana杂交工作站（Ventana Discovery，美国亚利桑那州图森市）中，在同一张微阵列芯片上完成共杂交。本数据集提交的数据为每个品系与每个时间点下3个生物学重复的平均值，共涵盖8组样本：gonad_resistant_date1、gonad_sensitive_date1、gonad_resistant_date2、gonad_sensitive_date2、gonad_resistant_date3、gonad_sensitive_date3、gonad_resistant_date4、gonad_sensitive_date4