|
| Status |
Public on May 01, 2014 |
| Title |
Blood male Saint Joseph Bay, FL SJB 2006_X24 |
| Sample type |
RNA |
| |
|
| Source name |
Peripheral Blood
|
| Organism |
Tursiops truncatus |
| Characteristics |
tissue: whole blood
gender: male
location: Saint Joseph Bay, FL
|
| Treatment protocol |
Blood samples were collected from wild dolphins in PAXgene™ Blood tubes (Qiagen, Valencia, CA, USA), mixed immediately to stabilize the RNA, and stored according to the manufacturer’s instructions.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA from blood samples from wild dolphins was extracted using the RNAeasy Kit according to the manufacturer (Qiagen, Valencia, CA). RNA was quantified using a NanoDrop ND-1000 (Wilmington, DE), qualified on an Agilent 2100 Bioanalyzer (Foster City, CA) and stored at −80°C until needed for gene expression profiling.
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 0.5 ug RNA using the One-Color Low RNA Input Amplification kit (Agilent) according to the manufacturer's instructions, followed by RNAeasy column purification (QIAGEN, Valencia, CA). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer and the Agilent 2100 Bioanalyzer.
|
| |
|
| Hybridization protocol |
500ng of Cy3-labelled cRNA was fragmented at 60°C for 30 minutes in a reaction volume of 250 ml containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 250 ml of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to custom Tursiops truncatus Microarray (Agilent) for 17 hours at 65°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute with 37°C GE Wash buffer 2 (Agilent), then dried immediately with a wash in acetonitrile (Agilent).
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner using one color scan setting for 4x44k array slides (Dye channel is set to Green and Green PMT is set to 100%).
|
| Description |
Gene expression wild dolphin blood
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software A8.5.3 (Agilent) using default parameters (protocol GE1) to obtain background subtracted and spatially detrended Processed Signal intensities. Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded.
|
| |
|
| Submission date |
Sep 06, 2013 |
| Last update date |
May 01, 2014 |
| Contact name |
Annalaura Mancia |
| E-mail(s) |
annalaura.mancia@unife.it
|
| Organization name |
University of Ferrara
|
| Department |
Life Sciences and Biotechnology
|
| Street address |
via L.Borsari, 46
|
| City |
Ferrara |
| ZIP/Postal code |
44121 |
| Country |
Italy |
| |
|
| Platform ID |
GPL17696 |
| Series (1) |
| GSE50684 |
Blood Transcriptome analysis of wild common bottlenose dolphins (Tursiops truncatus) from the South-East of the United States |
|
