|
| Status |
Public on Oct 15, 2007 |
| Title |
TNT_35ppm_Rep2 vs TNT_7ppm_Rep3 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
mRNA from worm (Rep#2) treated with 35 ppm of TNT labeled with Cyanine-3 (green)
|
| Organism |
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)
|
| 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 7 ppm of TNT labeled with Alex Fluor 647 (red)
|
| Organism |
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)
|
| 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 (1) its raw signal intensity was below its background level, (2) it overlapped with other spots, or (3) it was stained or over-saturated. The filtered spot intensity data were normalized by (1) subtraction of background intensity, (2) cross-channel LOWESS (local regression), and (3) centering to each channel's median spot intensity.
|
| |
|
| Submission date |
Feb 12, 2007 |
| Last update date |
Oct 02, 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 |
GPL4859 |
| Series (1) |
| GSE7024 |
Profiling differentially expressed earthworm genes in response to TNT exposure. |
|
