|
| Status |
Public on Oct 06, 2006 |
| Title |
Daphnia_pulex:Juvenile female vs Adult male, replicate 1 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Adult males
|
| Organism |
Daphnia pulex |
| Characteristics |
8 days old
|
| Treatment protocol |
Cultured at 20 degrees centigrade, in water from Lake Lemon, IN, filtered through 0.45 micron nylon, at 1 animal per 5 ml, fed Scenedesmus algae
|
| Growth protocol |
Cultured at 20 degrees centigrade, in water from Lake Lemon, IN, filtered through 0.45 micron nylon, at 1 animal per 5 ml, fed Scenedesmus algae
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Using Qiagen RNeasy Mini Kit reagents and protocol, except that Trizol was used for initial homogenization, and DNAse digestion was performed on-column.
|
| Label |
Cy5
|
| Label protocol |
20 micrograms total RNA reverse transribed into cDNA using Indirect cDNA Labeling Kit reagents (Invitrogen), according to manufacturer's protocol.
|
| |
|
| Channel 2 |
| Source name |
Juvenile females
|
| Organism |
Daphnia pulex |
| Characteristics |
6 days old
|
| Treatment protocol |
Cultured at 20 degrees centigrade, in water from Lake Lemon, IN, filtered through 0.45 micron nylon, at 1 animal per 5 ml, fed Scenedesmus algae
|
| Growth protocol |
Cultured at 20 degrees centigrade, in water from Lake Lemon, IN, filtered through 0.45 micron nylon, at 1 animal per 5 ml, fed Scenedesmus algae
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Using Qiagen RNeasy Mini Kit reagents and protocol, except that Trizol was used for initial homogenization, and DNAse digestion was performed on-column.
|
| Label |
Cy3
|
| Label protocol |
20 micrograms total RNA reverse transribed into cDNA using Indirect cDNA Labeling Kit reagents (Invitrogen), according to manufacturer's protocol.
|
| |
|
| |
| Hybridization protocol |
Equal masses of cDNA from males and females were combined in a 1:3 volume with hybridization buffer (50% formamide, 5X SSC, 0.1% SDS, 10 μg calf thymus DNA) and denatured at 90°C for 4 min, quick chilled, and injected into a Lucidea Slidepro automated hybridization chamber (Amersham) containing a microarray slide pre-hybridized according to manufacturer’s instructions. Following overnight hybridization, slides were washed twice at 60°C in 2X SSC + 0.2% SDS (15 min), once in 0.2X SSC + 0.2% SDS (10 min), once in 0.1X SSC (10 min) and once in 0.5X SSC (10 min). Slides were dipped briefly in 100% isopropanol, centrifuged 5 min at 500 x g to dry, and scanned using an Axon 4200B scanner.
|
| Scan protocol |
Fluorescent array images were collected for both Cy3 and Cy5 with a GenePix 4200B fluorescent scanner and image intensity data were extracted and analyzed with GenePix Pro 5.0 analysis software.
|
| Description |
Four biological replicates (double dye-swaps) were used to assess differences between males and juvenile females under standard laboratory culture conditions.
|
| Data processing |
Data were filtered and extracted using GenePix v. 5.0 and imported in Bioconductor. Normalization for M values was performed using spatial and intensity-dependent lowess smoothing in OLIN and differential expression was assessed using Limma.
|
| |
|
| Submission date |
Sep 25, 2006 |
| Last update date |
Oct 02, 2006 |
| Contact name |
John Kenneth Colbourne |
| E-mail(s) |
jcolbour@cgb.indiana.edu
|
| Phone |
(812) 856-0099
|
| Fax |
(812) 856-9340
|
| URL |
http://www.daphnia.cgb.indiana.edu
|
| Organization name |
Indiana University
|
| Department |
Biology
|
| Lab |
Center for Genomics and Bioinformatics
|
| Street address |
915 E. 3rd St.
|
| City |
Bloomington |
| State/province |
IN |
| ZIP/Postal code |
47405 |
| Country |
USA |
| |
|
| Platform ID |
GPL4349 |
| Series (1) |
| GSE5908 |
Profiling sex-biased and developmental gene expression in Daphnia pulex |
|
