|
| Status |
Public on Aug 27, 2008 |
| Title |
Earthworm exposed to RDX_Rep3 vs TNT_Rep3 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source Name |
mRNA from worm (Rep#3) treated with 30 ppm of RDX labeled with Cyanine-3 (green)
|
| Organism(s) |
Eisenia fetida |
| Characteristics |
Adult earthowrms exposed in soil for 28 days.
Worms were snap frozen in liquid nitrogen.
Worm tissue was fixed in RNAlater-ICE (Ambion).
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
RNeasy kit (Qiagen) and NucleoTrap Nucleic Acid Purification Kit (BD Biosciences)
|
| Label |
Cy3
|
| Label protocol |
30 ng mRNA was reverse-transcribed into cDNA and labeled using Genisphere 3DNA 900 Expression Array Detection Cy3 kit.
|
| |
|
| Channel 2 |
| Source Name |
mRNA from worm (Rep#3) treated with 50 ppm of TNT labeled with Alex Fluor 647 (red)
|
| Organism(s) |
Eisenia fetida |
| Characteristics |
Adult earthowrms exposed in soil for 28 days.
Worms were snap frozen in liquid nitrogen.
Worm tissue was fixed in RNAlater-ICe (Ambion).
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
RNeasy kit (Qiagen) and NucleoTrap Nucleic Acid Purification Kit (BD Biosciences)
|
| Label |
A647
|
| Label protocol |
30 ng mRNA was reverse transcribed into cDNA and labeled using Genisphere 3DNA 900 Expression Array Detection Alex Fluor 647 kit.
|
| |
|
| |
| Hybridization protocol |
Labeled cDNA probes for both channels (Genisphere) were added to the array, covered with a glass cover slip, which was then placed in a 50-ml tube. A few drops of water was also added into the tube to keep the air moisture. The capped tube was incubated at 55oC in a hybridization oven without rotation overnight (ca. 16 hr). Arrays were post-hyb washed according to manufacture's instruction (Genisphere).
|
| Scan protocol |
Arrays were scanned at 5-um resolution using VersArray ChipReader (Bio-Rad).
|
| Description |
A RNAlater-ICE fixed worm was chopped into 8-10 pieces in equal length. Total RNA was isolated using Qiagen RNeasy Mini Kit from each of the 8-10 pieces and was pooled as one biological replicate representing each individual earthworm. The pooled total RNA was purified to obtain mRNA using NucleoTrap Nucleic Acid Purification Kit (BD Biosciences).
|
| Data processing |
A spot was flagged out if its raw signal intensity was below its background level or the mean + 2x standard deviation of the 256 blank spots. The filtered spot intensity data were normalized by (1) subtraction of background intensity, and (2) centering to the mean spot signal intensity of each channel.
|
| |
|
| Submission date |
Aug 28, 2007 |
| 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 |
GPL5776 |
| Series (1) |
| GSE8909 |
Profiling differentially expressed earthworm genes in response to TNT, RDX, or TNT+RDX exposure. |
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