|
| Status |
Public on Jun 25, 2009 |
| Title |
fer-1 (hc1ts) mutant young adult biological rep 2 |
| Sample type |
RNA |
| |
|
| Source name |
adult worm
|
| Organism |
Caenorhabditis elegans |
| Characteristics |
tissue: whole worm
strain: BA1
genotype: fer-1 (hc1ts) mutant
|
| Treatment protocol |
Worms were washed from the plates with M9 solution and 1000 adult worms were collected into 1.5ml tubes using gating criteria on the COPAS Biosort that excluded L4 and younger animals. Immediately following sorting, worms were pelleted at 2,000 RPM and the supernatant was aspirated, leaving ~100µl on top of the worm pellet. 400µl of Trizol was added and the solution was vortexed for two minutes. Worms were stored at -80° until RNA isolation.
|
| Growth protocol |
Hypochlorite synchronized L1 stage wild type, fer-1(hc1), or fer-1(hc24) animals were grown to the young adult stage on standard NGM plates at 25°.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Trizol extraction of total RNA was performed according to the manufacturer's instructions.
|
| Label |
biotin
|
| Label protocol |
100ng of total RNA was converted to first-strand cDNA using reverse transcriptase primed by poly(T) and random oligomers that incorporated the T7 promoter sequence. Second-strand cDNA synthesis was followed by in vitro transcription with T7 RNA polymerase for linear amplification of each transcript, and the resulting cRNA was converted to cDNA, fragmented, assessed by Bioanalyzer, and biotinylated by terminal transferase end labeling.
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| |
|
| Hybridization protocol |
Labeled cDNA was added to Affymetrix hybridization cocktails, heated at 99ºC for 5 min and hybridized for 16 h at 45ºC to GPL200 GeneChips (Affymetrix Inc., Santa Clara CA). The microarrays were then washed at low (6X SSPE) and high (100mM MES, 0.1M NaCl) stringency and stained with streptavidin-phycoerythrin. Fluorescence was amplified by adding biotinylated anti-streptavidin and an additional aliquot of streptavidin-phycoerythrin stain.
|
| Scan protocol |
Affymetrix GCS3000 laser scanner was used to collect fluorescence signal after excitation at 570 nm.
|
| Description |
Gene expression data from young adult fer-1(hc1ts) mutant worms grown on 25ºC
|
| Data processing |
Affymetrix Command Console and Expression Console were used to quantitate expression levels for targeted genes; default values provided by Affymetrix were applied to all analysis parameters. Probes for each targeted gene were averaged and inter-array normalization performed using the GCRMA algorithm.
|
| |
|
| Submission date |
Jun 23, 2009 |
| Last update date |
Jun 24, 2009 |
| Contact name |
Predrag Krajacic |
| Organization name |
University of Pennsylvania
|
| Street address |
3700 Hamilton Walk A700 Richards
|
| City |
Philadelphia |
| State/province |
PA |
| ZIP/Postal code |
19104 |
| Country |
USA |
| |
|
| Platform ID |
GPL200 |
| Series (1) |
| GSE16753 |
C. elegans fer-1 mutant gene expression profile |
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