|
| Status |
Public on Apr 01, 2018 |
| Title |
300 ppb lufenuron 10155_Block4_updatedBlock4.txt |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Pool of approx. 500 copepodids (Atlantic Canada)
|
| Organism |
Lepeophtheirus salmonis |
| Characteristics |
treatment: 300 ppb lufenuron
|
| Treatment protocol |
Stock lufenuron (Elanco®) was prepared to 100 mg/L in 50% acetone, then diluted to working concentrations in seawater. Lufenuron dilutions were prepared to final concentrations of 0, 30, 300, 700, 1000, and 1500 parts per billion (n = 3 pools/treatment). Exposure time was 3 h before a 21 h holding in seawater. Pools of 500 copepodids were flash frozen post 24 h bioassay. treatment protocol
|
| Growth protocol |
Atlantic L. salmonis egg strings were obtained from adult female lice infecting Atlantic salmon in a salmon farm located in BMA-2a in the Bay of Fundy, New Brunswick. Eggs were hatched in a static sea water hatch system and grown to copepodids at Huntsman Marine Science Centre, St Andrew's, NB, Canada. growth protocol
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Lice were flash frozen in pools of 500 and kept at -80 degrees Celcius until RNA extraction (n = 21 pools). Total RNA was extracted from frozen tissue using TRIzol reagent® (Life Technologies), on-column DNase digestion (QIAGEN) followed by RNEasy column purification as per manufacturers’ instructions (QIAGEN). Purified total RNA was tested by agarose gel and automated electrophoresis (Experion; Bio-Rad) and spec. extract protocol
|
| Label |
Cy5-CTP
|
| Label protocol |
Labeled cRNA was generated from 825ng total RNA using Low-Input Quick Amp kits (Agilent; v6.5). A Cy3-cRNA reference pool to hybridize alongside samples was created for each of the above experiments, ensuring each experimental condition was included in the pool. label protocol
|
| |
|
| Channel 2 |
| Source name |
all condition pool
|
| Organism |
Lepeophtheirus salmonis |
| Characteristics |
sample type: reference
|
| Treatment protocol |
Stock lufenuron (Elanco®) was prepared to 100 mg/L in 50% acetone, then diluted to working concentrations in seawater. Lufenuron dilutions were prepared to final concentrations of 0, 30, 300, 700, 1000, and 1500 parts per billion (n = 3 pools/treatment). Exposure time was 3 h before a 21 h holding in seawater. Pools of 500 copepodids were flash frozen post 24 h bioassay. treatment protocol
|
| Growth protocol |
Atlantic L. salmonis egg strings were obtained from adult female lice infecting Atlantic salmon in a salmon farm located in BMA-2a in the Bay of Fundy, New Brunswick. Eggs were hatched in a static sea water hatch system and grown to copepodids at Huntsman Marine Science Centre, St Andrew's, NB, Canada. growth protocol
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Lice were flash frozen in pools of 500 and kept at -80 degrees Celcius until RNA extraction (n = 21 pools). Total RNA was extracted from frozen tissue using TRIzol reagent® (Life Technologies), on-column DNase digestion (QIAGEN) followed by RNEasy column purification as per manufacturers’ instructions (QIAGEN). Purified total RNA was tested by agarose gel and automated electrophoresis (Experion; Bio-Rad) and spec. extract protocol
|
| Label |
Cy3-CTP
|
| Label protocol |
Labeled cRNA was generated from 825ng total RNA using Low-Input Quick Amp kits (Agilent; v6.5). A Cy3-cRNA reference pool to hybridize alongside samples was created for each of the above experiments, ensuring each experimental condition was included in the pool. label protocol
|
| |
|
| |
| Hybridization protocol |
Samples were hybridized to oligonucleotide microarrays as per manufacturers' instructions for Low-Input Quick Amp kits (Agilent; v6.5). Arrays were designed using previously annotated ESTs from both Pacific and Atlantic L. salmonis (Yasuike et al. 2012; eArray design ID 024389; Agilent). hyb protocol
|
| Scan protocol |
Scanned at 5 micrometer resolution on a ScanArray Express (Perkin Elmer) and quantified on Imagene (v8.1; BioDiscovery). scan protocol
|
| Description |
300 ppb lufenuron
|
| Data processing |
For each probe on the microarray, the background median was subtracted from the foreground median. Data analysis was performed in GeneSpring GX11 (Agilent). Each array was normalized using an intensity-dependent Lowess normalization. All entities on the array are presented in the attached matrix file here, however, for the analysis, quality control filters for each of the three experiments retained probes that pass the following criteria in at least 65% of the samples in any one condition: raw signal ≥ 500 in both channels and no poor quality flags. data processing
|
| |
|
| Submission date |
Jun 09, 2017 |
| Last update date |
Apr 01, 2018 |
| Contact name |
Ben F Koop |
| E-mail(s) |
bkoop@uvic.ca
|
| Phone |
(250) 472-4067
|
| Organization name |
The University of Victoria
|
| Department |
Biology
|
| Lab |
Centre for Biomedical Research
|
| Street address |
PO Box 3020 STN CSC
|
| City |
Victoria |
| State/province |
BC |
| ZIP/Postal code |
V8W 3N5 |
| Country |
Canada |
| |
|
| Platform ID |
GPL15566 |
| Series (1) |
| GSE99880 |
High level efficacy of lufenuron against sea lice (Lepeophtheirus salmonis) linked to rapid impact on moulting processes |
|
