|
| Status |
Public on Feb 29, 2012 |
| Title |
Array 6_vitro_4-cell rep3 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Blastocyst cultured in vitro until 4-cell stage
|
| Organism |
Bos taurus |
| Characteristics |
sample type: in_vitro
breed (cattle): Simmental
reproductive status (cattle): Heifer
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA extraction was performed using the PicoPure RNA Isolation Kit (ARCTURS, Munich, Germany) according to the manufacturer's instruction.
|
| Label |
Cy5
|
| Label protocol |
Amplification of RNA has been done using the RiboAmpb HSPlus RNA Amplification kit (Molecular devices). This procedure may be required for microarray experiments with extremely limited amounts of starting material. The RiboAmpb HSPlus RNA Amplification Kit enables the production of microgram quantities of antisense RNA (aRNA) from picogram quantities of total cellular RNA. The RiboAmpb HSPlus Kit achieves high yields of aRNA with a proprietary linear amplification process using double-stranded cDNA as template in a two round T7 RNA polymerase catalyzed amplification.
Labelling aRNA was performed using the Universal Linkage System (ULS) by Kreatech for Gene Expression studies using microarray slides from Agilent. ULS is a non-enzymatic protocol that allows direct labeling of unmodified, amplified RNA. ULS will react with unmodified, amplified RNA and label it by formation of a coordinative bond on the N7 position of guanine. Fluorophore Cy3 and Cy5 are linked via a spacer to the ULS molecule.
|
| |
|
| Channel 2 |
| Source name |
in vivo control blastocyst
|
| Organism |
Bos taurus |
| Characteristics |
sample type: in_vivo
breed (cattle): Simmental
reproductive status (cattle): Heifer
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA extraction was performed using the PicoPure RNA Isolation Kit (ARCTURS, Munich, Germany) according to the manufacturer's instruction.
|
| Label |
Cy3
|
| Label protocol |
Amplification of RNA has been done using the RiboAmpb HSPlus RNA Amplification kit (Molecular devices). This procedure may be required for microarray experiments with extremely limited amounts of starting material. The RiboAmpb HSPlus RNA Amplification Kit enables the production of microgram quantities of antisense RNA (aRNA) from picogram quantities of total cellular RNA. The RiboAmpb HSPlus Kit achieves high yields of aRNA with a proprietary linear amplification process using double-stranded cDNA as template in a two round T7 RNA polymerase catalyzed amplification.
Labelling aRNA was performed using the Universal Linkage System (ULS) by Kreatech for Gene Expression studies using microarray slides from Agilent. ULS is a non-enzymatic protocol that allows direct labeling of unmodified, amplified RNA. ULS will react with unmodified, amplified RNA and label it by formation of a coordinative bond on the N7 position of guanine. Fluorophore Cy3 and Cy5 are linked via a spacer to the ULS molecule.
|
| |
|
| |
| Hybridization protocol |
A total of 825 ng of each labeled sample were incubated in a solution containing 2X blocking agent and 1X fragmentation buffer in a volume of 55 μl at 65 °C for 15 min, and were then put on ice. Fifty-five microliters of 2X GEx Hybridization Buffer HI-RPM was added for a final volume of 110 μl. The hybridization mix was added onto the array and hybridization was performed at 65 °C for 17 hr using an Agilent Hybridization chamber in a rotating oven
|
| Scan protocol |
Slides were scanned using Agilent’s High-Resolution C Scanner (Agilent Technologies, CA, USA) and features were extracted with the Agilent's Feature Extraction software (Agilent Technologies, CA, USA).
|
| Description |
Array 6
|
| Data processing |
Data were submitted to a simple background correction, a Loess within array normalization and a Quantile between array normalization.
|
| |
|
| Submission date |
Oct 28, 2011 |
| Last update date |
Feb 29, 2012 |
| Contact name |
Ahmed Gad |
| E-mail(s) |
[email protected]
|
| Organization name |
Colorado State University
|
| Department |
Biomedical Sciences
|
| Lab |
ARBL
|
| Street address |
3107 Rampart Rd
|
| City |
Fort Collins |
| State/province |
Colorado |
| ZIP/Postal code |
80521 |
| Country |
USA |
| |
|
| Platform ID |
GPL13226 |
| Series (1) |
| GSE33314 |
Gene expression analysis of bovine blastocyst produced under alternative in vivo and in vitro culture conditions during EGA time |
|
