|
| Status |
Public on May 12, 2014 |
| Title |
HLSV_30d_12dpi_rep3 |
| Sample type |
RNA |
| |
|
| Source name |
post 12 days HLSV inoculation
|
| Organism |
Nicotiana benthamiana |
| Characteristics |
tissue: upper new grown leaves
|
| Treatment protocol |
Twenty-four N. benthamiana plants were used for each batch of cross protection experiment between HLSV and TMV. When they were grown to 6 to 8 newly emerged and fully expanded leaves, 12 plants were inoculated with HLSV by mechanical inoculation. Two leaves on each plant were dusted with carborundum and inoculated with purified HLSV. Another 12 plants were treated with an equal volume of inoculation buffer. Among them, six plants were kept as a negative control and another six plants were used for TMV inoculation.
|
| Growth protocol |
N. benthamiana plants were grown in a growth room with a 16 h photoperiod and 8 h darkness, at a constant temperature of 23°C.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA extracted using Trizol following manufacturer's instructions.
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 0.5 ug RNA using the One-Color Low RNA Input Linear Amplification PLUS 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 |
1.5 ug 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 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 Agilent Whole Human Genome Oligo Microarrays (G4112A) 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 by brief centrifugation.
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2505B) using one color scan setting for 1x44k array slides (Scan Area 61x21.6 mm, Scan resolution 65 um, Dye channel is set to Green and Green PMT is set to 100%).
|
| Description |
Upper new grown leaf with HLSV inoculation, 30 d, 12 dpi, replicate 3
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 10.7 (Agilent) using default parameters (protocol GE1_107_Sep09 and Grid: 021113_D_F_20110323) to obtain background subtracted and spatially detrended Processed Signal intensities. Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded.
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| |
|
| Submission date |
May 22, 2013 |
| Last update date |
May 12, 2014 |
| Contact name |
Sek Man WONG |
| E-mail(s) |
dbswsm@nus.edu.sg
|
| Phone |
65-65162976
|
| Organization name |
National University of Singapore
|
| Department |
Biological sciences
|
| Lab |
Plant Molecular Virology
|
| Street address |
14 Science Drive 4
|
| City |
Singapore |
| State/province |
Singapore |
| ZIP/Postal code |
117543 |
| Country |
Singapore |
| |
|
| Platform ID |
GPL10098 |
| Series (1) |
| GSE47180 |
Profiling of genes related to cross protection and competition for NbTOM1 by HLSV and TMV |
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