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Status |
Public on Dec 04, 2013 |
Title |
LSS_liver_Skamania_rep3 |
Sample type |
RNA |
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Source name |
liver, wild fish, Skamania, replicate 3
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Organism |
Catostomus macrocheilus |
Characteristics |
tissue: liver Stage: reproductive adults collection site: Skamania
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Extracted molecule |
total RNA |
Extraction protocol |
Total RNA was extracted using STAT-60 (Tel-Test) following the manufacturer's instructions and RNA was resuspended in RNA secure (Ambion) to remove RNAse contamination. RNA was treated with TURBO DNA-free (Ambion) to remove DNA contamination and purified using RNeasy wcolumns (Qiagen). RNA was quantified using a NanoDrop-1000 spectrophotometer and quality was monitored with the Agilent 2100 Bioanalyzer (Agilent Technologies).
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Label |
Cy3
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Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 1000 ng RNA using the One-Color Microarray-Based Gene Expression Analysis (Quick Amp Labeling) kit from Agilent according to the manufacturer's instructions, followed by RNAeasy column purification (Qiagen). Cy3 dye incorporation and cRNA yield were measured using the NanoDrop ND-1000 Spectrophotometer.
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Hybridization protocol |
600 ng of Cy3-labelled cRNA (average specific activity =9.27 pmol Cy3/ug cRNA) was fragmented using the Agilent Gene Expression Hybridization kit and hybridized onto custom-designed fathead minnow (15K oligonulceotide) arrays for 17 hours at 65°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed for 1 min with GE Wash Buffer 1 (Agilent), GE Wash buffer 2 (Agilent) kept at 37°C , then 30 sec in Stabilization and Drying solution (Agilent).
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Scan protocol |
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2505B) using one color scan setting for 8x15k array slides (Scan Area 61x21.6 mm, Scan resolution 10um, Dye channel is set to Green and Green PMT is set to 100%).
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Description |
Gene expression profiling of wild largescale sucker from three sites in the Columbia River
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Data processing |
The scanned images were analyzed with Feature Extraction Software 9.1 (Agilent) using default parameters (protocol GE1-v5_10_Apr08 and Grid: 035137_D_F_20110628) to obtain background subtracted and spatially detrended Processed Signal intensities. Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded. Raw intensity data were log2 transformed.
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Submission date |
Sep 19, 2012 |
Last update date |
Dec 06, 2013 |
Contact name |
Alvine C Mehinto |
Organization name |
Southern California Coastal Water Research Project
|
Lab |
Center for Envirom & Human Toxicol
|
Street address |
3535 Harbor Blvd, Suite 110
|
City |
Costa Mesa |
ZIP/Postal code |
92626 |
Country |
USA |
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Platform ID |
GPL16082 |
Series (1) |
GSE41016 |
Microarray analysis of gene response to contaminant accumulation in wild largescale suckers |
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