|
| Status |
Public on May 10, 2012 |
| Title |
6 h auxin-treated seedlings vs control: replicate1 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
6 h auxin-treated seedlings
|
| Organism |
Arabidopsis thaliana |
| Characteristics |
strain: Col. 0
age: day 14
tissue: seedling
agent: auxin
|
| Treatment protocol |
After 14 d in the growth chamber, seedlings were removed from the agar and equilibrated for 1 h at 22 °C in 20 ml deionized water, and then transferred to 20 ml of deionized water containing 20 µM auxin and 0.0125% dimethyl sulfoxide, and then incubated under light. Plants were collected at 0 (control) and 6 h, and immediately frozen in liquid nitrogen, and then stored at –80 °C before analysis.
|
| Growth protocol |
The sterilized seeds were placed on 0.8% agar (Nakalai Tesque, Inc., Kyoto, Japan) with Murashige and Skoog Plant Salt Mixture (Nihon Pharmaceutical Co., Ltd., Tokyo, Japan) and 1% sucrose (Wako Pure Chemical Industries, Osaka, Japan) in a sterilized Petri dish (90-mm diameter). The seeds were held at 4 °C overnight in darkness and then transferred to a growth chamber. Emerging plants were maintained in the growth chamber under a schedule of 16-h light (22 °C) and 8-h dark (20 °C).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA extracted using RNeasy following manufacturer's instructions
|
| Label |
Cy-5
|
| Label protocol |
Two-color spike mix was added to the total RNA, and the RNA was labeled with a Quick Amp Labeling Kit, two-color (Agilent Technologies), according to the manufacturer’s protocol. Fluorescent cRNA was generated from total RNA: 500 ng of RNA was reverse-transcribed with MMLV reverse transcriptase and an oligo(dT) primer containing the T7 promoter, and subsequently transcribed in vitro using T7 RNA polymerase, resulting in Cy3-labeled (control) and Cy5-labeled (cis-CA-treated) cRNAs. The cRNAs were purified through RNeasy Mini Spin columns (Qiagen) and then quantified with a NanoDrop ND-1000 UV-VIS spectrophotometer
|
| |
|
| Channel 2 |
| Source name |
control seedlings
|
| Organism |
Arabidopsis thaliana |
| Characteristics |
strain: Col. 0
age: day 14
tissue: seedling
agent: control
|
| Treatment protocol |
After 14 d in the growth chamber, seedlings were removed from the agar and equilibrated for 1 h at 22 °C in 20 ml deionized water, and then transferred to 20 ml of deionized water containing 20 µM auxin and 0.0125% dimethyl sulfoxide, and then incubated under light. Plants were collected at 0 (control) and 6 h, and immediately frozen in liquid nitrogen, and then stored at –80 °C before analysis.
|
| Growth protocol |
The sterilized seeds were placed on 0.8% agar (Nakalai Tesque, Inc., Kyoto, Japan) with Murashige and Skoog Plant Salt Mixture (Nihon Pharmaceutical Co., Ltd., Tokyo, Japan) and 1% sucrose (Wako Pure Chemical Industries, Osaka, Japan) in a sterilized Petri dish (90-mm diameter). The seeds were held at 4 °C overnight in darkness and then transferred to a growth chamber. Emerging plants were maintained in the growth chamber under a schedule of 16-h light (22 °C) and 8-h dark (20 °C).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA extracted using RNeasy following manufacturer's instructions
|
| Label |
Cy-3
|
| Label protocol |
Two-color spike mix was added to the total RNA, and the RNA was labeled with a Quick Amp Labeling Kit, two-color (Agilent Technologies), according to the manufacturer’s protocol. Fluorescent cRNA was generated from total RNA: 500 ng of RNA was reverse-transcribed with MMLV reverse transcriptase and an oligo(dT) primer containing the T7 promoter, and subsequently transcribed in vitro using T7 RNA polymerase, resulting in Cy3-labeled (control) and Cy5-labeled (cis-CA-treated) cRNAs. The cRNAs were purified through RNeasy Mini Spin columns (Qiagen) and then quantified with a NanoDrop ND-1000 UV-VIS spectrophotometer
|
| |
|
| |
| Hybridization protocol |
Mixtures of 0.825 µg of Cy3-labeled and Cy5-labeled cRNAs were co-hybridized at 65 °C for 17 h on an Agilent Technologies 4 × 44K Arabidopsis 4 60-mer oligo-microarray.
|
| Scan protocol |
The slides were then washed and scanned with an Agilent G2505B Scanner to detect fluorescence intensity.
|
| Description |
Biological replicate 1 of 2
|
| Data processing |
Agilent Feature Extraction Software (v 8.5.1.1) was used for background subtraction and LOWESS normalization. Data represent the log ratio (treated/control).
|
| |
|
| Submission date |
May 10, 2012 |
| Last update date |
May 10, 2012 |
| Contact name |
Naoya Wasano |
| E-mail(s) |
wasano@cc.tuat.ac.jp
|
| Organization name |
Tokyo University of Agriculture & Technology
|
| Street address |
Saiwai-cho 3-5-8
|
| City |
Fuchu |
| State/province |
Tokyo |
| ZIP/Postal code |
183-8509 |
| Country |
Japan |
| |
|
| Platform ID |
GPL9020 |
| Series (1) |
| GSE37899 |
Arabidopsis thaliana: auxin-treated seedlings versus control seedlings |
|
