|
| Status |
Public on Nov 09, 2015 |
| Title |
cad_1_mixtox65_Cy3 |
| Sample type |
RNA |
| |
|
| Source name |
total RNA from 30 animals, cad_1
|
| Organism |
Folsomia candida |
| Characteristics |
developmental stage: adult
growth medium: LUFA2.2 control soil
|
| Treatment protocol |
Total RNA was extracted from all animals, using the SV Total RNA isolation System (Promega).
|
| Growth protocol |
Approximately 30 adults individuals were exposed in glass vials containing ~30 g of soil at 50% of the soil WHC. Exposure was done at same conditions as incubation. After exposure the animals were extracted from soil with tap water and snap frozen in liquid N2
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cyanine-3 (Cy3) or Cyanine-5 (Cy5) labeled cRNA was prepared from 500 ng total RNA using the Low Input Quick Amp Labeling Kit, two-color (Agilent Technologies) according to the manufacturer's guidelines followed by RNAeasy column purification (QIAGEN). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.
|
| Label |
Cy3
|
| Label protocol |
825 ng of Cy3-labeled or Cy5-labeled cRNA (specific activity >8.0 pmol dye/ug cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 55 ul containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturer's instructions. On completion of the fragmentation reaction, 55 ul of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Custom Gene Expression Microarrays with 4x180k format for 17 hours at 65°C in a rotating Agilent hybridization oven (10 rpm) After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute with 37°C GE Wash buffer 2 (Agilent), washed in acetonitril for 10 seconds, then dried immediately by air.
|
| |
|
| Hybridization protocol |
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2505B) using two color scan setting for 4x180k array slides (Scan Area 61x21.6 mm, Scan resolution 5um, Dye channel is set to Red & Green and PMT is set to 100%.
|
| Scan protocol |
The scanned images were analyzed with Feature Extraction Software 9.5.1.1 (Agilent) using default parameters (protocol GE2_107_Sep09 and Grid: 042279_D_F_20120723) to obtain mean Red or Green intensities and median Red or Green background intensities. In limma, edwards method for background correction was used (offset = 30), and global loess normalizing was done.
|
| Description |
raw data: mixtox65.txt: Cy3
X7
used in analysis: YES
|
| Data processing |
background corrected (edwards method, min. offset at 30) loess normalized, log transformed intensities
|
| |
|
| Submission date |
Jul 14, 2015 |
| Last update date |
Nov 09, 2015 |
| Contact name |
Tjalf de Boer |
| E-mail(s) |
[email protected]
|
| Organization name |
MicroLife Solutions
|
| Street address |
Science Park 406
|
| City |
Amsterdam |
| ZIP/Postal code |
1098 XH |
| Country |
Netherlands |
| |
|
| Platform ID |
GPL7150 |
| Series (1) |
| GSE70877 |
Combined transcriptomics classifier analysis reveals a set of adverse effect genes for use as potential endpoint in effect based screening |
|
