|
| Status |
Public on Nov 12, 2009 |
| Title |
CW (15-048) vs. OSU (15-028) on male embryos 15 dpf Array #034 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Male CW embryo on 15dpf (sample#: 15-048)
|
| Organism |
Oncorhynchus mykiss |
| Characteristics |
Male embryo on 15 dpf (sample#: 15-048), possessing CW allele (fast-developing allele) on rainbow trout embryonic development rate QTL
|
| Treatment protocol |
A fourth generation backcross (BC4) family was made at Washington State University, using marker assisted selection to select for third-generation backcross (BC3) individuals possessing the allele for fast development (from the Clearwater (CW) clonal line) for crossing with the Oregon State University (OSU) rainbow trout clonal line, which possesses alleles for slower development at the major embryonic development rate QTL (Nichols et al., 2007). Briefly, sperm from an XY BC3 male with the OSU/CW genotype at the major embryonic development rate QTL was utilized to fertilize eggs from an OSU female at Washington State University. Backcross individuals were sampled at 15 days, 19 days and 28 days post-fertilization (dpf), which roughly correspond to the development stages of eyed and yolk vascularizaiton, caudal flexing and beginning of hatching, respectively. After fertilization, embryos were incubated in re-circulating stack egg incubators in a constant 11ºC climate-controlled room. Genetic markers tightly linked to the major embryonic development rate QTL were used to identify the genotype of each embryo.
|
| Growth protocol |
After fertilization, embryos were incubated in re-circulating stack egg incubators in a constant 11ºC climate-controlled room before sample collection.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from each frozen rainbow trout embryo using TRI reagent followed by RNeasy kit (Qiagen) purification.
|
| Label |
Cy3
|
| Label protocol |
Invitrogen Indirect cDNA Labeling System version 3 (http://web.uvic.ca/grasp/microarray/array.html)
|
| |
|
| Channel 2 |
| Source name |
Male OSU embryo on 15dpf (sample#: 15-028)
|
| Organism |
Oncorhynchus mykiss |
| Characteristics |
Male embryo on 15 dpf (sample#: 15-028), possessing OSU allele (slow-developing allele) on rainbow trout embryonic development rate QTL
|
| Treatment protocol |
A fourth generation backcross (BC4) family was made at Washington State University, using marker assisted selection to select for third-generation backcross (BC3) individuals possessing the allele for fast development (from the Clearwater (CW) clonal line) for crossing with the Oregon State University (OSU) rainbow trout clonal line, which possesses alleles for slower development at the major embryonic development rate QTL (Nichols et al., 2007). Briefly, sperm from an XY BC3 male with the OSU/CW genotype at the major embryonic development rate QTL was utilized to fertilize eggs from an OSU female at Washington State University. Backcross individuals were sampled at 15 days, 19 days and 28 days post-fertilization (dpf), which roughly correspond to the development stages of eyed and yolk vascularizaiton, caudal flexing and beginning of hatching, respectively. After fertilization, embryos were incubated in re-circulating stack egg incubators in a constant 11ºC climate-controlled room. Genetic markers tightly linked to the major embryonic development rate QTL were used to identify the genotype of each embryo.
|
| Growth protocol |
After fertilization, embryos were incubated in re-circulating stack egg incubators in a constant 11ºC climate-controlled room before sample collection.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from each frozen rainbow trout embryo using TRI reagent followed by RNeasy kit (Qiagen) purification.
|
| Label |
Cy5
|
| Label protocol |
Invitrogen Indirect cDNA Labeling System version 3 (http://web.uvic.ca/grasp/microarray/array.html)
|
| |
|
| |
| Hybridization protocol |
Invitrogen Indirect cDNA Labeling System version 3 (http://web.uvic.ca/grasp/microarray/array.html)
|
| Scan protocol |
Following hybridization and slide washing, microarray signals were detected with the Scanarray 4000 Microarray Scanner (Perkin Elmer, Waltham, MA) at excitation wavelengths 540 nm for Cy3 and 633 nm for Cy5, 10 µm resolution and laser power settings 70 – 90%, photo multiplier tube (PMT) gain 80-90%.
|
| Description |
CW (15-048) vs. OSU (15-028) on male embryos 15 dpf
|
| Data processing |
background values of each spot were obtained from the ImaGene image segmentation algorithms. Each spot was flagged for quality with flags for “good spot”, “empty spot”, or “poor spot”. Raw data from ImaGene were collated for data analysis in SAS and SAS JMP Genomics (SAS Institute, Cary, NC). Prior to data analysis, local background-subtracted signals for each spot on each slide were natural log-transformed and then standardized by subtracting the median value of all the spots except “poor” spots flagged by ImaGene.
|
| |
|
| Submission date |
Nov 12, 2008 |
| Last update date |
Nov 17, 2008 |
| Contact name |
Peng Xu |
| E-mail(s) |
[email protected]
|
| Phone |
7654961030
|
| Organization name |
Purdue University
|
| Department |
Biology
|
| Lab |
Nichols Lab
|
| Street address |
915 W State St
|
| City |
West Lafayette |
| State/province |
IN |
| ZIP/Postal code |
47907 |
| Country |
USA |
| |
|
| Platform ID |
GPL2716 |
| Series (1) |
| GSE13570 |
Transcriptome profiling of embryonic development rate in rainbow trout advance backcross introgression lines |
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