|
| Status |
Public on Feb 02, 2012 |
| Title |
SN comparison_11dpi_rep3 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
plants infected with PVX/HCLH at 11dpi
|
| Organism |
Nicotiana benthamiana |
| Characteristics |
tissue: symptomatic, non-inoculated upper leaves
|
| Treatment protocol |
Samples (pooled systemic leaves) were collected at 7 or 11 days postinoculation (SN comparison), at 4 or 8 days after infiltration (VIGS Prot), and at 24 or 72 hours after temperature shift (SHR). 4 week old plants were inoculated with PVX/HCLH by rubbing sap from PVX/HCLH infected plants
|
| Growth protocol |
Grown in soil in growth chamber using 16 h period of light (25±2°C) and 8 h of darkness (22±1°C), light intensity 70-90 mmol m-2 s-2 , 65±2 % humidity
|
| Extracted molecule |
total RNA |
| Extraction protocol |
For each biological replicate, two symptomatic, non-inoculated upper leaves from each of ten plants per treatment were harvested and immediately frozen in liquid N2. Total RNA was extracted from plant leaves using Trizol reagent, followed by purification with RNeasy midiprep columns(Qiagen)
|
| Label |
Cy3
|
| Label protocol |
RNA was amplified with the MessageAmp II aRNA amplification kit (Ambion, Inc, Austin, USA) as described in the instruction manual. Aminoallyl UTP was added to the mix of the T7 RNA polymerase-driven aRNA amplification reaction. The amount of aRNA was quantified with a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies). aRNA quality was assessed with a 2100 Bioanalyzer (Agilent Technologies). 5 ug of aminoallyl-labeled aRNA were resuspended in 0.1 M Na2CO3 (pH 9.0) and labeled with Hyper5 and Cy3 Mono NHS Ester (CyTMDye Post-labeling Reactive Dye Pack, Amersham). Samples were purified following the manufacturer's instructions for Megaclear TM (Ambion) and Hyper5 and Cy3 incorporation was measured using 1 ul of the probe in the Nanodrop
|
| |
|
| Channel 2 |
| Source name |
plants infected with PVX/HCWT at 11dpi
|
| Organism |
Nicotiana benthamiana |
| Characteristics |
tissue: symptomatic, non-inoculated upper leaves
|
| Treatment protocol |
Samples (pooled systemic leaves) were collected at 7 or 11 days postinoculation (SN comparison), at 4 or 8 days after infiltration (VIGS Prot), and at 24 or 72 hours after temperature shift (SHR). 4 week old plants were inoculated with PVX/HCWT by rubbing sap from PVX/HCWT infected plants.
|
| Growth protocol |
Grown in soil in growth chamber using 16 h period of light (25±2°C) and 8 h of darkness (22±1°C), light intensity 70-90 mmol m-2 s-2 , 65±2 % humidity
|
| Extracted molecule |
total RNA |
| Extraction protocol |
For each biological replicate, two symptomatic, non-inoculated upper leaves from each of ten plants per treatment were harvested and immediately frozen in liquid N2. Total RNA was extracted from plant leaves using Trizol reagent, followed by purification with RNeasy midiprep columns(Qiagen)
|
| Label |
Cy5
|
| Label protocol |
RNA was amplified with the MessageAmp II aRNA amplification kit (Ambion, Inc, Austin, USA) as described in the instruction manual. Aminoallyl UTP was added to the mix of the T7 RNA polymerase-driven aRNA amplification reaction. The amount of aRNA was quantified with a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies). aRNA quality was assessed with a 2100 Bioanalyzer (Agilent Technologies). 5 ug of aminoallyl-labeled aRNA were resuspended in 0.1 M Na2CO3 (pH 9.0) and labeled with Hyper5 and Cy3 Mono NHS Ester (CyTMDye Post-labeling Reactive Dye Pack, Amersham). Samples were purified following the manufacturer's instructions for Megaclear TM (Ambion) and Hyper5 and Cy3 incorporation was measured using 1 ul of the probe in the Nanodrop
|
| |
|
| |
| Hybridization protocol |
The hybridization experiment was performed according to the manufacture's protocol (Agilent technologies, Agilent 60-mer oligo microarray processing protocol: Two color microarray based gene expression analysis, G4140-90050)
|
| Scan protocol |
The slides were scanned with a GenePix 4000B scanner and the images were quantified with GenePix Pro 5.1 software (Molecular Devices).
|
| Description |
PVX/HCWT_vs_PVX/HCLH_11dpi_rep3
|
| Data processing |
Data processing was carried-out using the non-parametric algorithm Rank Products (Breitling et al. 2004) and available as RankProd package at Bioconductor (Hong et al. 2006). Raw intensities were background-substracted by NORMEXP method with a offset of 50. Signals (in log2 scale) were then normalized by LOWESS algorithm (intra-arrays normalization) followed by adjustment of their quantiles (inter-arrays normalization).
|
| |
|
| Submission date |
Jan 04, 2012 |
| Last update date |
Feb 02, 2012 |
| Contact name |
Francisco Tenllado |
| Organization name |
Centro de Investigaciones Biológicas, CSIC
|
| Department |
Plant Biology
|
| Lab |
Plant Molecular Virology
|
| Street address |
Ramiro de Maeztu, 9
|
| City |
Madrid |
| State/province |
Madrid |
| ZIP/Postal code |
28006 |
| Country |
Spain |
| |
|
| Platform ID |
GPL10098 |
| Series (1) |
| GSE34841 |
Comparative transcriptomic responses to compatible and incompatible plant-virus interactions that lead to cell death |
|
