|
| Status |
Public on Jan 01, 2012 |
| Title |
Daphnia pulex POVI_15 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
whole animal, Lake Murray
|
| Organism |
Daphnia pulex |
| Characteristics |
gender: female
age: 11 d
|
| Treatment protocol |
We collected epilimnetic lakewater from Lake Murray, brought it back to the lab and screened it through an 80 um mesh to remove any zooplankton. This lakewater therefore contained the seston that forms the natural resource environment for zooplankton. We conducted a 24-hr pulse experiment where half of the individuals, selected at random, were transferred into a beaker containing screened lakewater from Lake Murray; these were not fed A. falcatus that day. The remaining beakers were transferred to filtered water from Lake Murray and fed A. falcatus at a density of 23,000 cells ml-1. Thus the only difference between the Lake Murray seston treatment and the A. falcatus control was natural seston vs. lab food
|
| Growth protocol |
Beakers of experimental animals were randomly distributed within an environmental chamber at 20°C on a 12:12 L:D cycle. Experimental animals were fed a satiating level (at least 20,000 cells/ml) of the standard lab diet, vitamin-enriched A. falcatus, daily. Animals were transferred to new beakers thrice a week until the experimental animals were 10 d old.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
total RNA was extracted from frozen tissue using a Qiagen RNeasy kit with the optional on-column DNase treatment
|
| Label |
Alexafluor 555
|
| Label protocol |
1.5 µg of each sample was amplified and labeled using the Amino Allyl MessageAmp II aRNA Amplification kit (Ambion) according to the manufacturer recommendations. 8 µg of amplified RNA was labeled with either Alexa Fluor 555 or Alexa Fluor 647 dyes (Invitrogen).
|
| |
|
| Channel 2 |
| Source name |
whole animal, Ankistrodesmus
|
| Organism |
Daphnia pulex |
| Characteristics |
gender: female
age: 11 d
|
| Treatment protocol |
We collected epilimnetic lakewater from Lake Murray, brought it back to the lab and screened it through an 80 um mesh to remove any zooplankton. This lakewater therefore contained the seston that forms the natural resource environment for zooplankton. We conducted a 24-hr pulse experiment where half of the individuals, selected at random, were transferred into a beaker containing screened lakewater from Lake Murray; these were not fed A. falcatus that day. The remaining beakers were transferred to filtered water from Lake Murray and fed A. falcatus at a density of 23,000 cells ml-1. Thus the only difference between the Lake Murray seston treatment and the A. falcatus control was natural seston vs. lab food
|
| Growth protocol |
Beakers of experimental animals were randomly distributed within an environmental chamber at 20°C on a 12:12 L:D cycle. Experimental animals were fed a satiating level (at least 20,000 cells/ml) of the standard lab diet, vitamin-enriched A. falcatus, daily. Animals were transferred to new beakers thrice a week until the experimental animals were 10 d old.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
total RNA was extracted from frozen tissue using a Qiagen RNeasy kit with the optional on-column DNase treatment
|
| Label |
Alexafluor 647
|
| Label protocol |
1.5 µg of each sample was amplified and labeled using the Amino Allyl MessageAmp II aRNA Amplification kit (Ambion) according to the manufacturer recommendations. 8 µg of amplified RNA was labeled with either Alexa Fluor 555 or Alexa Fluor 647 dyes (Invitrogen).
|
| |
|
| |
| Hybridization protocol |
1 µg of each of the two paired labeled samples was hybridized at 42°C during 16 hours to microarrays. We used MAUI SC mixers, a 4-bay MAUI Hybridization system and Pronto Universal Microarray Hybridization kits (Corning) according to the manufacturer recommendations. Array washes after hybridizations were performed using the Pronto kit.
|
| Scan protocol |
slides were scanned on a Perkin Elmer ProScanArray Express HT scanner and the images quantified using Perkin Elmer ScanArray Express SP3 software
|
| Data processing |
All arrays were assayed for data quality, and normalized using print-tip loess without background correction because nearly all spot-background correlations were below 0.2. Between-slide normalization was done with the Aquantile method. All data processing was done in the limma package of R.
|
| |
|
| Submission date |
Jul 27, 2011 |
| Last update date |
Jan 01, 2012 |
| Contact name |
Jeffry L. Dudycha |
| E-mail(s) |
dudycha@biol.sc.edu
|
| Organization name |
University of South Carolina
|
| Department |
Biological Sciences
|
| Street address |
715 Sumter Street
|
| City |
Columbia |
| State/province |
SC |
| ZIP/Postal code |
29208 |
| Country |
USA |
| |
|
| Platform ID |
GPL13280 |
| Series (1) |
| GSE31001 |
Daphnia ecotypes fed lab versus natural resources |
|
