|
| Status |
Public on May 08, 2008 |
| Title |
Liver_Ciguatoxin (P-CTX-1)_24h_052+ |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Liver from mice exposed to 1% Tween 60 in physiological saline (vehicle)
|
| Organism |
Mus musculus |
| Characteristics |
Adult male C57-BL6 mice with frequency transmitters implanted in the abdominal cavity
|
| Treatment protocol |
Three groups of control mice (n=3) were injected ip with a single dose of physiological saline with 1% Tween 60 (vehicle). Three groups of experimental mice (n=3) were injected ip with a single dose of 0.26 ng/g Pacific Ciguatoxin-1 (P-CTX-1) in vehicle. At 1, 4, and 24 h post-injection, 3 experimental mice and 3 time-matched controls were anesthetized with 50 mg/mL sodium pentobarbital ip. Livers were immediately dissected, flash frozen in liquid nitrogen, and stored at -80 °C until RNA processing.
|
| Growth protocol |
Adult male C57-BL6 mice were maintained on a 12 h:12 h light:dark cycle and were given food and water ad libitum. Radio frequency transmitters were implanted in the abdominal cavity of the mice to measure core temperature and motor activity. The mice were allowed at least 10 days of recovery before testing while animal health was monitored.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
The livers of the 3 control mice for each time point were pooled prior to RNA extraction while RNA was extracted from livers of individual CTX exposed mice. The livers were crushed using a BioPulverizer (BioSpec Products, Inc.) in liquid nitrogen, immediately placed in cooled Tri-Reagent (Molecular Research Center, Inc.) and homogenized using a Tissue-Tearor (United Lab Plastics). All homogenates were processed according to the manufacturer’s protocol. RNA was suspended in nuclease-free water and cleaned using an RNeasy midi-column (Qiagen). RNA was quantified using a NanoDrop ND-1000 and qualified on an Agilent 2100 Bioanalyzer.
|
| Label |
Cy3
|
| Label protocol |
Four hundred fifty nanograms of total RNA from control and experimental animals was separately amplified and labeled with either Cy3 or Cy5 labeled CTP (Perkin Elmer) using Agilent’s Low Input Linear Amplification kit according to manufacturer’s protocol. Following labeling and clean up, amplified RNA and dye incorporation were quantified using a NanoDrop ND-1000.
|
| |
|
| Channel 2 |
| Source name |
Liver from mouse exposed to 0.26 ng/g Pacific Ciguatoxin-1
|
| Organism |
Mus musculus |
| Characteristics |
Adult male C57-BL6 mice with frequency transmitters implanted in the abdominal cavity
|
| Treatment protocol |
Three groups of control mice (n=3) were injected ip with a single dose of physiological saline with 1% Tween 60 (vehicle). Three groups of experimental mice (n=3) were injected ip with a single dose of 0.26 ng/g Pacific Ciguatoxin-1 (P-CTX-1) in vehicle. At 1, 4, and 24 h post-injection, 3 experimental mice and 3 time-matched controls were anesthetized with 50 mg/mL sodium pentobarbital ip. Livers were immediately dissected, flash frozen in liquid nitrogen, and stored at -80 °C until RNA processing.
|
| Growth protocol |
Adult male C57-BL6 mice were maintained on a 12 h:12 h light:dark cycle and were given food and water ad libitum. Radio frequency transmitters were implanted in the abdominal cavity of the mice to measure core temperature and motor activity. The mice were allowed at least 10 days of recovery before testing while animal health was monitored.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
The livers of the 3 control mice for each time point were pooled prior to RNA extraction while RNA was extracted from livers of individual CTX exposed mice. The livers were crushed using a BioPulverizer (BioSpec Products, Inc.) in liquid nitrogen, immediately placed in cooled Tri-Reagent (Molecular Research Center, Inc.) and homogenized using a Tissue-Tearor (United Lab Plastics). All homogenates were processed according to the manufacturer’s protocol. RNA was suspended in nuclease-free water and cleaned using an RNeasy midi-column (Qiagen). RNA was quantified using a NanoDrop ND-1000 and qualified on an Agilent 2100 Bioanalyzer.
|
| Label |
Cy5
|
| Label protocol |
Four hundred fifty nanograms of total RNA from control and experimental animals was separately amplified and labeled with either Cy3 or Cy5 labeled CTP (Perkin Elmer) using Agilent’s Low Input Linear Amplification kit according to manufacturer’s protocol. Following labeling and clean up, amplified RNA and dye incorporation were quantified using a NanoDrop ND-1000.
|
| |
|
| |
| Hybridization protocol |
One microgram each of Cy3 and Cy5 labeled targets were combined and hybridized to an Agilent catalog 44K whole genome mouse oligonucleotide array for 17 h at 60 °C in an Agilent DNA Microarray Hybridization Oven. Arrays were then washed consecutively in solutions of 6X SSPE with 0.005% N-lauroylsarcosine and 0.06X SSPE with 0.005% N-Lauroylsarcosine for 1 m each at room temperature. This was followed by a final 30 s wash in Agilent Stabilization and Drying solution.
|
| Scan protocol |
Microarrays were imaged on an Agilent microarray scanner at 10µm scan resolution. Concurrent scans of the red and green PMT were performed.
|
| Description |
24 h exposure to 0.26 ng/g Pacific Ciguatoxin-1
|
| Data processing |
Agilent Feature Extraction software version A8.5.1 using Agilent Design File 012694_D_G220051010.
|
| |
|
| Submission date |
Mar 09, 2008 |
| Last update date |
May 08, 2008 |
| Contact name |
Frances M Van Dolah |
| E-mail(s) |
fran.vandolah@noaa.gov
|
| Organization name |
Marine Biotoxins Program, NOAA Center for Coastal Environmental Health and Biomolecular Research
|
| Street address |
219 Fort Johnson Rd
|
| City |
Charleston |
| State/province |
SC |
| ZIP/Postal code |
29412 |
| Country |
USA |
| |
|
| Platform ID |
GPL2872 |
| Series (1) |
| GSE10768 |
Liver Genomic Responses to Ciguatoxin (P-CTX-1) |
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