|
| Status |
Public on Sep 14, 2007 |
| Title |
AGILENT-SiIn-B |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Infloresence - mixed stage, immature buds, Second Replica
|
| Organism |
Arabidopsis thaliana |
| Characteristics |
Plants were grown in soil in growth chamber with 16 hours of light for 5 weeks. Immature inflorescence was harvested ~2 hours after dark. Tissue included inflorescence meristem and early stage floral buds (up to Stage 11/12). Total RNA was isolated using TRIzol reagent.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated using TRIzol reagent
|
| Label |
Cy3
|
| Label protocol |
Prior to hybridizations, the quality and quantity of the total RNA sample was confirmed by running 10ng samples on an Agilent bioanalyser (RNA chip), and by using a spectrophotometer. The oligoarray hybridization experiments included six technical replicates, of which three were dye-reversal. Five hundred ng of total RNA was used as template for cRNA production and Cyanine dyes was incorporated using the Agilent low RNA input linear amp kit (5198-3523; Agilent). Normal yields from 500 ng total RNA input using a 4 h in vitro transcription were 15 ug cRNA (15 pmole cyanine dye incorporated/ug cRNA).
|
| |
|
| Channel 2 |
| Source name |
Silique - 24 to 48 hr post-fertilization, Second Replica
|
| Organism |
Arabidopsis thaliana |
| Characteristics |
Plants were grown in soil in growth chamber with 16 hours of light for 5 weeks. Siliques were harvested ~24-48 hours after fertilization, when petals have begun to detach (Stage 16-17). Tissue was harvested ~2 hours after dark. Length of siliques was 5-10 mm. Total RNA was isolated using TRIzol reagent.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated using TRIzol reagent
|
| Label |
Cy5
|
| Label protocol |
Prior to hybridizations, the quality and quantity of the total RNA sample was confirmed by running 10ng samples on an Agilent bioanalyser (RNA chip), and by using a spectrophotometer. The oligoarray hybridization experiments included six technical replicates, of which three were dye-reversal. Five hundred ng of total RNA was used as template for cRNA production and Cyanine dyes was incorporated using the Agilent low RNA input linear amp kit (5198-3523; Agilent). Normal yields from 500 ng total RNA input using a 4 h in vitro transcription were 15 ug cRNA (15 pmole cyanine dye incorporated/ug cRNA).
|
| |
|
| |
| Hybridization protocol |
One ug of labeled cRNA (cy3 and cy5 labeled sample) was diluted to 175 µl and defragmented at 60°C for 30 min following the Agilent hybridization protocols (5184-3568; Agilent). Defragmented samples were diluted with concentrated hybridization buffer to 500 µl (30% formamide final concentration) as described in Hughes et al (2001) and hybridized for 20 h at 40°C using Agilent hybridization chambers.
|
| Scan protocol |
Arrays were washed and dried as described in Hughes et al (2001) and scanned on an Agilent G2565BA microarray scanner (Coughlan et al. 2004).
|
| Description |
RNA extraction in Meyers Lab (http://mpss.udel.edu/). Agilent Experiment by Sean J Coughlan from Agilent (http://www.agilent.com/).
|
| Data processing |
The intensities of Cy3- and Cy5-labeled probes were normalized by comparing signal intensities of house keeping genes (positive controls) for both dyes and using the determined ratio as a correction factor for differences in labeling efficiencies(Agilent Feature Extraction v8.1 software). The genes that had valid signal in all six replicates were exported to Rosetta Resolver software, Seattle, Washington). The normalized values were used to calculate the ratio of channel intensities (Cy5/Cy3), which were then log10 transformed.
|
| |
|
| Submission date |
Sep 10, 2007 |
| Last update date |
Aug 14, 2011 |
| Contact name |
Junfeng Chen |
| E-mail(s) |
[email protected]
|
| Organization name |
University of Manchester
|
| Department |
School of Computer Science
|
| Street address |
Kilburn Building, Oxford Road
|
| City |
Manchester |
| ZIP/Postal code |
M13 9PL |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL888 |
| Series (1) |
| GSE8994 |
A Comparison of microarray and MPSS Technology Platforms for Expression Analysis of Arabidopsis |
|
