|
| Status |
Public on Oct 05, 2013 |
| Title |
low_DDVP-continuous_expose-26h_harvest-rep3 |
| Sample type |
RNA |
| |
|
| Source name |
whole organism
|
| Organism |
Caenorhabditis elegans |
| Characteristics |
developmental stage: L4 larvae
exposure concentration: 0.6 µM DDVP (low)
exposure protocol duration: continuous
harvest time: 26
|
| Treatment protocol |
Cultures of 250,000 synchronized L1 worms were grown in 30 mL CeHR medium. A separate T-75 culture flask was set up for each condition. After ~41 h, when 50% of the worms had passed the L3/L4 molt, the exposures were initiated. Four flasks of worms were harvested prior to beginning the exposure as the 0 h controls. Equal volumes of water or dichlorvos stock (diluted for 0.6 µM or 15 µM final concentration as appropriate) were added to the remaining flasks, which were then returned to the incubator. The flasks were treated according to one of three protocols. In the first protocol, the set of flasks was incubated without interruption for the duration of the experiment (continuous) with flasks being harvested at 2, 8, 14, 20, and 26 h. The other two sets were incubated for 2 or 8 h, at which time the worms were centrifuged out of the exposure medium, washed 3 times with a modified CeHR medium (Washout Buffer), resuspended in fresh CeHR medium without dichlorvos, and returned to the incubator. Flasks were harvested at 6 h intervals following the washout through 26 h. Untreated controls (or shams) and low and high concentrations flasks were prepared for each harvest time of each of the three protocols.
|
| Growth protocol |
C. elegans cultures were grown in CeHR medium at 22.5 °C in T-75 tissue culture flasks with shaking at 70 rpm.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Flash frozen worms were pulverized under liquid nitrogen in a Spex 6750 Freezer Mill. RNA was extracted from pulverized worms with Trizol followed by an additional purification step using RNeasy Midi columns. PolyA RNA was then isolated using OligoTex. All steps were performed following manufacturers recommendations.
|
| Label |
Biotin
|
| Label protocol |
cDNA was synthesized using SuperScript Choice kit and a T24T7 primer following the manufacturer's protocol. Biotin labeled cRNA was synthesized using the Enzo BioArray High Yield Kit following the manufacturer's protocol.
|
| |
|
| Hybridization protocol |
Biotin labeled cRNA was fragmented and hybridized to Affymetrix C. elegans whole genome GeneChips as recommended by Affymetrix.
|
| Scan protocol |
Hybridized Affymetrix C. elegans whole genome GeneChips were then scanned as recommended by Affymetrix.
|
| Description |
gene expression data from L4 larvae
|
| Data processing |
Probe set expression levels were calculated using RMA processing of raw data within Partek Genomics Suite (Partek, Inc.).
|
| |
|
| Submission date |
Oct 05, 2012 |
| Last update date |
Oct 05, 2013 |
| Contact name |
John A. Lewis |
| E-mail(s) |
john.a.lewis1@us.army.mil
|
| URL |
http://usacehr.amedd.army.mil
|
| Organization name |
U.S. Army Center for Environmental Health Research
|
| Department |
Biomarkers Program
|
| Street address |
568 Doughten Drive
|
| City |
Ft. Detrick |
| State/province |
MD |
| ZIP/Postal code |
21702-5010 |
| Country |
USA |
| |
|
| Platform ID |
GPL200 |
| Series (1) |
| GSE41366 |
Alterations in gene expression in Caenorhabditis elegans associated with organophosphate pesticide intoxication and recovery |
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