|
| Status |
Public on Oct 14, 2009 |
| Title |
Treated Worm-1 6 day (PMT 350) |
| Sample type |
RNA |
| |
|
| Source name |
whole organism
|
| Organism |
Eisenia fetida |
| Characteristics |
protocol: 6 days treatment
agent: CL-20
tissue: whole organism
|
| Treatment protocol |
Adults bearing clitelum and weighing 0.4~0.5 g were pulled out of the culture and allowed to depurate overnight prior to being exposed to CL-20 on filter paper (0.2 ug/cm2). Worms were snap-frozen upon experimental termination.
|
| Growth protocol |
Earthworms were kept in moistened sphagnum peat (pH 6.5–7.5, moisture content 50%) and fed ad libitum on a diet of with Magic Worm Food (Carolina Biological Supply).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from RNAlater-ICE fixed worms using the RNeasy mini kit (QIAGEN, Valencia, CA) following the manufacturer's recommendations. RNA was quantified using a NanoDrop-1000 spectrophotometer and quality was monitored with the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA).
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 0.5 ug RNA using the One-Color Low RNA Input Linear Amplification PLUS kit (Agilent) according to the manufacturer's instructions, followed by RNAeasy column purification (QIAGEN, Valencia, CA). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.
|
| |
|
| Hybridization protocol |
600 ug of Cy3-labelled cRNA (specific activity >9.0 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 25 ml containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 25 ml of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to the custom-designed Agilent test array for 17 hours at 65°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent), 1 minute with 37°C GE Wash buffer 2 (Agilent), 30 seconds with Acetonitrile, and dried with Agilent Stabilization and Drying Solution.
|
| Scan protocol |
Slides were scanned immediately after washing on GenePix 4200AL scanner at PMT gain level 350/450/510 using default setting (Scan resolution 5um, One channel-Green, Laser power 100%).
|
| Description |
Gene expression profiling
barcode: 251642310001
Array#_SLide#: A4_SL1
PMT: 350
|
| Data processing |
The scanned images were analyzed with GenePix Pro 6.1 (Molecular Devices, Sunnyvale, CA) using default parameters and Agilient-provided GAL grid file 016423_D_20070410.gal to obtain signal intensities. Non-uniform spots and spots with a signal intensity outside the linear range were flagged. Signal intensity was converted into relative RNA concentration based on the linear standard curve of spike-in RNAs. The relative RNA concentration was normalized to the median value on each array.
|
| |
|
| Submission date |
Oct 13, 2009 |
| Last update date |
Oct 13, 2009 |
| Contact name |
Ping Gong |
| E-mail(s) |
ping.gong@us.army.mil
|
| Phone |
(601) 634-3521
|
| Organization name |
US Army ERDC
|
| Department |
Environmental Laboratory
|
| Lab |
Environmental Genomics and Genetics
|
| Street address |
3909 Halls Ferry Road
|
| City |
Vicksburg |
| State/province |
MS |
| ZIP/Postal code |
39180 |
| Country |
USA |
| |
|
| Platform ID |
GPL9416 |
| Series (1) |
| GSE18536 |
Microarray analysis of CL-20 reversible neurotoxicity in the earthworm Eisenia fetida |
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