|
| Status |
Public on Apr 16, 2012 |
| Title |
VH Soil EC50 rep2 |
| Sample type |
RNA |
| |
|
| Source name |
total RNA from 10 animals
|
| Organism |
Folsomia candida |
| Characteristics |
soil: LUFA soil with vlagheide soil
concentration: EC50
|
| Treatment protocol |
Approximately 10 animals, 23 d old, were exposed for four days in glass jars containing ~25 g of soil (both field soils and control soils at 50% water holding capacity) Exposure was done at same conditions as incubation. After exposure the animals were extracted from soil with tap water via floatation, put in 1.5 mL tubes and frozen in liquid N2.
|
| Growth protocol |
Cultures were first age synchronized before exposure to soil: 20 adult animals were incubated per container; containing a plaster of Paris base mixed with charcoal, to lay eggs for 2 days. The incubation conditions were 20°C, 75% RH and 12:12 hour dark:light cycle
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from all animals (~30) per jar, using SV Total RNA isolation System (Promega).
|
| Label |
cy5
|
| Label protocol |
Cyanine-3 (Cy3) or Cyanine-5 (Cy5) labeled cRNA was prepared from 500 ng total RNA using the Quick Amp Labeling Kit, two-color (Agilent Technologies) according to the manufacturer's guidelines with a slight modification where the transcription (labeling) reactions were done in half the volume, followed by RNAeasy column purification (QIAGEN). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.
|
| |
|
| Hybridization protocol |
300 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 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 Custom Gene Expression Microarrays with 8 - 15 k format (GPL7150) 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.
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2505B) using two color scan setting for 8x15k array slides (Scan Area 61x21.6 mm, Scan resolution 5um, Dye channel is set to Red&Green and PMT is set to 100%, XDR Hi 100%, XDR Lo 10%).
|
| Description |
Raw data file: Sarray02.txt
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 9.5.1.1 (Agilent) using default parameters (protocol GE2-v5_95 and Grid: 016342_D_F_20070323) to obtain mean Red or Green intensities and median Red or Green background intesities. In limma, edwards method for background correction was used (offset = 30), and global loess normalizion (MAANOVA) was done.
|
| |
|
| Submission date |
Apr 11, 2012 |
| Last update date |
Apr 16, 2012 |
| Contact name |
Dick Roelofs |
| E-mail(s) |
[email protected]
|
| Phone |
+31-20-5987078
|
| Fax |
+31-20-5987123
|
| URL |
http://www.falw.vu.nl/nl/onderzoek/ecological-sciences/animal-ecology/staff/dick-roelofs.asp
|
| Organization name |
VU University
|
| Department |
Animal Ecology
|
| Lab |
Roelofs' lab
|
| Street address |
De Boelelaan 1085
|
| City |
Amsterdam |
| State/province |
Noord-Holland |
| ZIP/Postal code |
1081HV |
| Country |
Netherlands |
| |
|
| Platform ID |
GPL7150 |
| Series (1) |
| GSE37154 |
Functional environmental genomics of a municipal landfill soil |
|
