|
| Status |
Public on Feb 15, 2019 |
| Title |
L-Thr-treatment_36h_rep1 |
| Sample type |
RNA |
| |
|
| Source name |
mycellium at 36 h after L-Thr-treatment
|
| Organism |
Fusarium graminearum |
| Characteristics |
strain: JCM 9873
treatment: L-Thr-treatment
time after spraying: 36 h
tissue: mycellium
|
| Treatment protocol |
The L-Thr solution was added to brown rice flour medium after autoclave sterilization and mixed well before turning into a hard mass by cooling at room temperature. The treated medium was immediately used for inoculation of conidia. After incubating at 25°C in a sealed container for 36 or 72 hrs, the culture was used for RNA isolation.
|
| Growth protocol |
Fusarium graminearum strain JCM9873 was cultured on brown rice flour medium with or without L-Thr treatment at 25°C. Mycelia were grown for 36 or 72 hrs on the medium.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Mycellium were ground in liquid nitrogen. Total RNA was prepared using a Qiagen Plant RNA Isolation Kit.
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 200 ng RNA using the Low RNA Input Quick Amp 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.
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| |
|
| Hybridization protocol |
600 ng of Cy3-labelled cRNA (specific activity >10.0 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 25 ul containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 55 ul of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent Custum Fusarium graminearum Microarray 8x15K 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 by raise it slowly from buffer 2.
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2565BA) using one color scan setting for 4x44k array slides (Scan Area 61x21.6 mm, Scan resolution 5um, Dye channel is set to Green. Scan mode is eXtended Dynamic range Scan mode and PMT is set to 10% and 100%).
|
| Description |
L-Thr_36h_rep1
|
| Data processing |
The images were analyzed using Feature Extraction Software (Ver. 10.7.3.1; Agilent Technologies). These data were analyzed further with GeneSpring GX12.0 software (Agilent Technologies). Normalization was performed as follows: 1, intensity-dependent Lowess normalization; 2, data transformation, with measurements less than 0.01 set to 0.01; 3, per-chip normalization, in which the 75th percentile method was used to normalize each array
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| |
|
| Submission date |
Jun 12, 2018 |
| Last update date |
Feb 15, 2019 |
| Contact name |
Takumi Nishiuchi |
| E-mail(s) |
tnish9@staff.kanazawa-u.ac.jp
|
| Organization name |
Kanazawa University
|
| Street address |
13-1 Takaramachi
|
| City |
Kanazawa |
| ZIP/Postal code |
920-0934 |
| Country |
Japan |
| |
|
| Platform ID |
GPL25174 |
| Series (1) |
| GSE115673 |
Global gene expression analysis of supression of mycotoxin biosyntheies by L-Thr in Fusarium graminearum. |
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