Unknown,Transcriptomics,Genomics,Proteomics

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Toxicogenomic analyses of Ruditapes philippinarum by DNA microarray: new tools for anthropogenic impact assessment on the Venice Lagoon.

ABSTRACT: A manila clam oligo microarray platform (GPL10900) was used to profile gene expression in digestive gland of R. philippinarum sampled in four seasons in 4 different areas of Venice Lagoon. For each tissue, total RNA was extracted from four (4) independent biological replicates of digestive gland, each consisting of tissue pools of five (5) animals. In this study, we analyzed 64 samples (pools of 5 digestive gland). Gene expression profiling was performed using the Agilent-027304 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.

ORGANISM(S): Ruditapes philippinarum

SUBMITTER: Massimo Milan

PROVIDER: E-GEOD-26985 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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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: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.