|
| Status |
Public on Jul 05, 2011 |
| Title |
After Individual 5 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Arctic charr gill tissue whole RNA reverse transcribed to cDNA, immediately after cooling
|
| Organism |
Salvelinus alpinus |
| Characteristics |
tissue: gill
condition: immediately after cooling from moderate temperature stress
individual: 5
|
| Treatment protocol |
200 Arctic charr were exposed to chronic, moderate heat stress (15-19ºC) for 72 hrs. 10 fish were sampled before (control), during (after 72 hrs of exposure) and after (12 hrs after returning to ambient temperature) heat exposure, then after 72 hrs of recovery time, representing the C, D, A and R treatment groups, respectively. Six randomly sampled fish from each treatment group were used for microarray analysis.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated from gill tissues disrupted and homogenized in 1 ml TRIzol reagent using a Mixer-mill (Retch® MM 301) with tungsten carbide beads. Phase separation was conducted using 200 µl chloroform, and RNA was purified using the RNeasy Mini Kit (Qiagen) following the manufacturer’s instructions.
|
| Label |
Cy5
|
| Label protocol |
cDNA (300 ng) and aRNA (500 ng) were labeled with Cy5 and Cy3 (Amersham Biosciences), respectively, using Invitrogen’s SuperScript Indirect cDNA Labeling System following the manufacturer's instructions.
|
| |
|
| Channel 2 |
| Source name |
reference aRNA
|
| Organism |
Salmo salar |
| Characteristics |
tissue: gonad, brain, spleen
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated from gill tissues disrupted and homogenized in 1 ml TRIzol reagent using a Mixer-mill (Retch® MM 301) with tungsten carbide beads. Phase separation was conducted using 200 µl chloroform, and RNA was purified using the RNeasy Mini Kit (Qiagen) following the manufacturer’s instructions.
|
| Label |
Cy3
|
| Label protocol |
cDNA (300 ng) and aRNA (500 ng) were labeled with Cy5 and Cy3 (Amersham Biosciences), respectively, using Invitrogen’s SuperScript Indirect cDNA Labeling System following the manufacturer's instructions.
|
| |
|
| |
| Hybridization protocol |
Details of the microarray hybridization process can be found at the University of Victoria cGRASP website (http://web.uvic.ca/grasp/microarray/array.html) within the .pdf document entitled Invitrogen Indirect cDNA Labeling System version 3.
|
| Scan protocol |
Images were scanned on a ScanArray™ Express Microarray Scanner (Packard BioScience BioChip Technologies, model #ASCEX00).
Slides were scanned at 74 and 72 PMT for Cy3 and Cy5, respectively, and spot intensity was calculated with ImaGene ver. 6.5.1.
|
| Description |
A5
|
| Data processing |
All statistical analyses of the microarray data were conducted using Genespring ver. 7.3.1. Signals were normalized per spot and per chip using an intensity-dependent (LOWESS) normalization, then per gene to normalize to the median. Spots were filtered on flags present, and only spots with signals greater or equal to the average base/proportional value of the raw channel were retained.
|
| |
|
| Submission date |
May 27, 2011 |
| Last update date |
Jul 05, 2011 |
| Contact name |
Nicole Lisa Quinn |
| E-mail(s) |
nicole_quinn@sfu.ca
|
| Organization name |
Simon Fraser University
|
| Department |
Molecular Biology and Biochemistry
|
| Lab |
Davidson, SSB 6150
|
| Street address |
8888 University Drive
|
| City |
Burnaby |
| State/province |
BC |
| ZIP/Postal code |
V5A 1S6 |
| Country |
Canada |
| |
|
| Platform ID |
GPL10096 |
| Series (1) |
| GSE29610 |
Identification of genes expressed during and following exposure to chronic moderate temperature stress in Arctic charr |
|
