Unknown,Transcriptomics,Genomics,Proteomics

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A microarrays-based analysis of gametogenesis in two Portuguese populations of the European clam Ruditapes decussatus

ABSTRACT: The European clam, Ruditapes decussatus (Linnaeus, 1758) is a bivalve mollusc of the family Veneridae native to the European Atlantic and Mediterranean coastal waters. Its production is exclusively based on natural recruitment, which is subject to high annual fluctuations due to adversely affected by pollution and other environmental factors. Microarray analyses have been performed in four gonadal maturation stages of two higly productive Portuguese wild populations (Ria Formosa in South and Ria de Aveiro in North) characterized by different responses to spawning induction. Twenty clams (Ruditapes decussatus)were monthly sampled between July 2011 and July 2012 in two different Portuguese wild populations: Ria Formosa and Ria de Aveiro. At each sampling date the gonads of the collected clams were immediately dissected for RNA extraction and fixed for histological analysis. Total RNA was isolated using Extract-all (Eurobio) procedure. RNA quality and integrity was controlled on the Agilent bioanalyzer using RNA nanochips and Agilent RNA 6000 nanoreagents (Agilent Technologies, Waldbronn, Germany). RNA concentrations were measured at 260 nm using a ND-1000 spectrophotometer (Nanodrop Technologies) using the conversion factor 1 OD = 40 mg/mL total RNA. Samples were stored at -80°C until further use. Gene expression profiling was performed using an R.decussatus oligo-DNA microarray of 59,951 probes based on single-colour detection (Cyanine-3 only). Microarrays were scanned with Agilent scanner G2565BA at a resolution of 2 microns; all slides were scanned twice at two different sensitivity settings (XDRHi 100% and XDRLo 10%); the scanner software created a unique ID for each pair of XDR scans and saved it to both scan image files. FeatureExtraction v10.7.3.1 used XDR ID to link the pairs of scans together automatically when extracting data. The signal left after all the FE processing steps have been completed is ProcessedSignal that contains the Multiplicatively Detrended, Background-Subtracted Signal.

ORGANISM(S): Ruditapes decussatus

SUBMITTER: Massimo Milan

PROVIDER: E-GEOD-51150 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:The Manila clam (Ruditapes philippinarum) is a cultured bivalve species with high worldwide commercial importance. Nevertheless, diseases can cause high economical losses. For this reason, the study of immune genes in bivalve mollusks has increased in the last years. The present work describes the construction of the first R. philippinarum microarray containing immune-related hemocyte sequences and its application for the study of the gene transcription profiles of hemocytes from clams challenged with Vibrio alginolyticus through a time course. A comparative analysis of gene expression was conducted between R. philippinarum infected and non-infected by V. alginolyticus clam hemocytes. Clams (n=100) were notched in the shell next to the adductor muscles and injected with 100 Âµl of Vibrio alginolyticus, strain TA15, (10^8 UFC/ml in PBS) to mimic an intramuscular infection. Controls (n=100) were injected with 100 Âµl of PBS. After stimulation, clams were returned to the tanks and maintained at 15ÂºC until sampling at 3, 8, 24, and 72 hours after challenge Hemolymph (1 ml) was withdrawn from the adductor muscle of the clams with a 0.5mm diameter (25G) disposable needle. Hemolymph from four individuals was pooled and biological replicates were taken at each sampling point. Hemolymph was centrifuged at 4Â°C at 3000 g for 10 minutes. The pellet was resuspended in 250 Âµl of Trizol (Invitrogen). Total RNA isolation was conducted following the manufacturer's specifications in combination with the RNeasy mini kit (Qiagen) for RNA purification after DNase I treatment. Gene expression profiling was performed using an R. philippinarum oligo-DNA microarray of 13,671 probes based on single-colour detection (Cyanine-3 only). Microarrays were scanned with Agilent scanner G2565BA at a resolution of 5 microns; all slides were scanned twice at two different sensitivity settings (XDRHi 100% and XDRLo 10%); the scanner software created a unique ID for each pair of XDR scans and saved it to both scan image files. Feature Extraction (FE) 9.5 used XDR ID to link the pairs of scans together automatically when extracting data. The signal left after all the FE processing steps have been completed is ProcessedSignal that contains the Multiplicatively Detrended, Background-Subtracted Signal.

Project description:Digestive Gland Samples: A manila clam oligo microarray platform (GPL10900) was used to profile gene expression in digestive gland of R. philippinarum. Total RNA was extracted from three (3) independent biological replicates of digestive gland for each sampling site, each consisting of tissue pools of five (5) animals. Statistical analysis with SAM (Significance Analysis of Microarray) identified1,127 probes differentially expressed. Gills Samples: A manila clam oligo microarray platform (GPL10900) was used to profile gene expression in gills of R. philippinarum. Total RNA was extracted from three (3) independent biological replicates of gills for each sampling site, each consisting of tissue pools of five (5) animals. Statistical analysis with SAM (Significance Analysis of Microarray) identified1,127 probes differentially expressed. Digestive Gland Samples: In this study, we analyzed six (6) samples, three (3) pools of digestive gland of Manila clam sampled in Marghera and three(3) pools of digestive gland of Manila clam sampled in Alberoni. Gene expression profiling was performed using the Agilent-019810 Ruditapes philippinarum Oligo Microarray platform (GPL10900) based on single-colour detection (Cyanine-3 only). Microarrays were scanned with Agilent scanner G2565BA (barcode on the left, DNA on the back surface, scanned through the glass) at a resolution of 5 microns; all slides were scanned twice at two different sensitivity settings (XDRHi 100% and XDRLo 10%); the scanner software created a unique ID for each pair of XDR scans and saved it to both scan image files. Feature Extraction (FE) 9.5 used XDR ID to link the pairs of scans together automatically when extracting data. The signal left after all the FE processing steps have been completed is ProcessedSignal that contains the Multiplicatively Detrended, Background-Subtracted Signal. Gills Samples: In this study, we analyzed six (6) samples, three (3) pools of gills of Manila clam sampled in Marghera and three(3) pools of gills of Manila clam sampled in Alberoni. Gene expression profiling was performed using the Agilent-019810 Ruditapes philippinarum Oligo Microarray platform (GPL10900) based on single-colour detection (Cyanine-3 only). Microarrays were scanned with Agilent scanner G2565BA (barcode on the left, DNA on the back surface, scanned through the glass) at a resolution of 5 microns; all slides were scanned twice at two different sensitivity settings (XDRHi 100% and XDRLo 10%); the scanner software created a unique ID for each pair of XDR scans and saved it to both scan image files. Feature Extraction (FE) 9.5 used XDR ID to link the pairs of scans together automatically when extracting data. The signal left after all the FE processing steps have been completed is ProcessedSignal that contains the Multiplicatively Detrended, Background-Subtracted Signal.

Project description:A C. gallina microarray platform was developed to assess variations on transcritpomic profiles between different seasons and sampling sites A comparative analysis of gene expression was conducted in Chamelea gallina collected in two different areas along Abruzzi coasts subjected to different mortality events. In particular sampling activities have been performed in collaboration with fishing cooperatives along Abruzzi coasts, Chamelea gallina clams have been collected from two sites (T4 and T7) at 0.25 mi (ca. 0.4 km) at commercial size (25 mm) and harvested by hydraulic dredge. The harvesting has been conducted in three periods of 2014 (July, August and September). The time of sampling has been obliged by a dramatic reduction of harvesting in T4 that has been reported by fishing cooperatives during spring. After sampling, clams have been kept with ice till their arrival in laboratory, within 6 hours. From each sampling site and each period, the digestive gland of 30 clams has been dissected and frozen at -21Â°C, for a total of 150 clams divided in 5 pools for each period and each site of sampling. During the clam sampling, from the same sites, the upper layer of bed sediments (0â10 cm) was collected by a small grab sampler in July and September. Composite sediment samples have been refrigerated during transport and stored at â 20 Â°C within a few hours from sampling, until analysis. Briefly, about 400 g of sediment have been suitably homogenized and dried in an oven at 40 Â° C for 24 hours, then crushed, ground and sieved to 2 mm. A portion of about 100 g for each sample has been further ground and screened to 0.2 mm for heavy metal analysis. The content of cadmium (Cd), lead (Pb) and arsenic (As) have been performed, after extraction by aqua regia, by Inductively Coupled Plasma-Optical Emission Spectrometer ( ICP-OESâ¦..) the analysis have been recorded as means triplicate measurements. The determination of Hg have been done in Atomic Adsorption with Hydride System. Ten grams (accuracy Â± 0.0001 g) of dry and homogenized sediment of 0.2 mm has been put into a clean centrifuge tube, and a 1:1 (v/v) acetone/n-hexane (5 mL), and surrogate standard mixture (2-fluorobiphenyl and 4-terphenyl-d14) solutions were then added, after extraction and clean-up processes, the samples were analyzed with a gas chromatographerâelectron capture detector (GM-ECD) to evaluate concentrations of Polycyclic aromatic hydrocarbons (PAHs) and organophosphate pesticides (OPPs). Organochloride pesticides (OCPs) have been analysed, after purification, with static headspace GC Gene expression profiling was performed using an Chamelea gallina-specific oligo-DNA microarray of15,019 probes representing 12,064 transcripts based on single-colour detection (Cyanine-3 only). Microarrays were scanned with Agilent scanner G2565BA (barcode on the left, DNA on the back surface, scanned through the glass) at a resolution of 5 microns; all slides were scanned twice at two different sensitivity settings (XDRHi 100% and XDRLo 10%); the scanner software created a unique ID for each pair of XDR scans and saved it to both scan image files. Feature Extraction (FE) 9.5 used XDR ID to link the pairs of scans together automatically when extracting data. The signal left after all the FE processing steps have been completed is ProcessedSignal that contains the Multiplicatively Detrended, Background-Subtracted Signal.

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A microarrays-based analysis of gametogenesis in two Portuguese populations of the European clam Ruditapes decussatus

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