Mycroarray_Hypoxia_Oyster

Marine intertidal organisms commonly face hypoxic stress during low tide emersion; moreover, eutrophic conditions and sediment nearness could lead to hypoxic phenomena; it is indeed important to understand the molecular processes involved in the response to hypoxia. In this study the molecular response of the Pacific oyster Crassostrea gigas to prolonged hypoxia (2 mg O2 L-1 for 20 d) was investigated under experimental conditions. A transcriptomic approach was employed using a cDNA microarray of 9058 C. gigas clones to highlight the genetic expression patterns of the Pacific oyster under hypoxic conditions. Lines of oysters resistant (R) and susceptible (S) to summer mortality were used in this study. This is the first study employing microarrays to characterize the genetic markers and metabolic pathways responding to hypoxic stress in C. gigas. For the microarray analysis oysters were sampled at days 0, 2, 10, and 20 after the beginning of hypoxia. On each date, the digestive gland from 6 oysters was dissected, pooled, and stored at -80°C in Extract-All Reagent (Eurobio) at a concentration of 1 mL/50 mg tissue until total RNA was extracted. For each condition (hypoxia and normoxia) and oyster line (R and S), 4 pools were sampled (biological replicates) for a total of 56 pools during the 4 days of sampling. Furthermore, the entire tissue from 10 wild oysters was collected, pooled, and homogenized in Extract-all Reagent (Eurobio) to constitute a single total RNA sample to use as a reference in all slide hybridizations and Real-Time PCR analyses. Five μg of total RNA were directly labeled by reverse transcription using the Direct ChipShot Direct Labeling and Clean-Up Kit (Promega). The samples were labeled with Cyanine-5 (Cy5). The reference samples were labeled with Cyanine-3 (Cy3) in separate tubes following the same protocol. The Cy3-labeled cDNAs were pooled and re-divided to obtain a homogeneous reference sample. cDNA samples and references were evaporated in a SpeedVac and mixed into a single pool in equimolar amounts with the Chip Hybe hybridization buffer (Ventana Discovery). Hybridization was performed using a Ventana automatic hybridization station (Ventana Discovery).