|
| Status |
Public on Aug 24, 2009 |
| Title |
smoke-butenolide 24h, rep 2 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
smoke-water treated maize kernel, 24h after germination
|
| Organism |
Zea mays |
| Characteristics |
strain: Mv255
tissue: Embryo
age: 24h
|
| Treatment protocol |
The batches of kernels were submerged into 20 ml water (control), 1:1000 (v/v) dilution of smoke-water and 0,1 µM butenolide.
|
| Growth protocol |
The kernels were decontaminated in 3% sodium hypochlorite containing Tween 20 and 70% EtOH (10 min each). For germination, each treatments consisted of six biological replicates (15 kernels in each), and seeds were germinated in a controlled environmental chamber (25 °C, 100 µmol m–2 s–1 light intensity).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
For RNA isolation from control and smoke (1:1000) and butenolide-treated kernels, identical conditions were applied as for the germination tests and embryos were harvested 1,5, 3, 6,9,12, and 24 h after treatment. At 24h, only the kernels with ruptured testa were selected for further experiments. Total RNA was isolated from the maize kernel embryo axes (without scutellum) using TRIzol reagent (Invitrogen) and cleaned up with Qiagen RNeasy Plant Mini Kit (Qiagen) applying a few modifications. RNA was then treated with RNase-free DNase I (Qiagen) according to the manufacturer’s instructions. The RNA Integrity Number (RIN) of the samples was determined using the Agilent BioAnalyzer. Only samples with a RIN ≥ 8 were considered for further analysis.
|
| Label |
Cy3
|
| Label protocol |
400 ng total RNA was amplified and aminoallyl-UTP was incorporated using 101 TargetAmp Kit (Epicentre) and the resulting aaRNA was labelled with Cy3 and Cy5 (Amersham).
|
| |
|
| Channel 2 |
| Source name |
butenolide-water treated maize kernel, 24h after germination
|
| Organism |
Zea mays |
| Characteristics |
tissue: Embryo
age: 24h
strain: Mv255
|
| Treatment protocol |
The batches of kernels were submerged into 20 ml water (control), 1:1000 (v/v) dilution of smoke-water and 0,1 µM butenolide.
|
| Growth protocol |
The kernels were decontaminated in 3% sodium hypochlorite containing Tween 20 and 70% EtOH (10 min each). For germination, each treatments consisted of six biological replicates (15 kernels in each), and seeds were germinated in a controlled environmental chamber (25 °C, 100 µmol m–2 s–1 light intensity).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
For RNA isolation from control and smoke (1:1000) and butenolide-treated kernels, identical conditions were applied as for the germination tests and embryos were harvested 1,5, 3, 6,9,12, and 24 h after treatment. At 24h, only the kernels with ruptured testa were selected for further experiments. Total RNA was isolated from the maize kernel embryo axes (without scutellum) using TRIzol reagent (Invitrogen) and cleaned up with Qiagen RNeasy Plant Mini Kit (Qiagen) applying a few modifications. RNA was then treated with RNase-free DNase I (Qiagen) according to the manufacturer’s instructions. The RNA Integrity Number (RIN) of the samples was determined using the Agilent BioAnalyzer. Only samples with a RIN ≥ 8 were considered for further analysis.
|
| Label |
Cy5
|
| Label protocol |
400 ng total RNA was amplified and aminoallyl-UTP was incorporated using 101 TargetAmp Kit (Epicentre) and the resulting aaRNA was labelled with Cy3 and Cy5 (Amersham).
|
| |
|
| |
| Hybridization protocol |
The dye-labelled probes were cleaned up (Qiagen), mixed with the corresponding samples, concentrated, resuspended in the hybridization solution containing formamide and incubated at 42 °C overnight in a hybridization oven. Finally, slides were washed with different concentrations of SSC at room temperature.
|
| Scan protocol |
Scanning was performed using an Amersham Typhoon Trio+ scanner with default settings. The detection of signal intensities and the grid adjustment were accomplished with ArrayVision software version 8.0 (Amersham). The intensity value of each spot and background region, multiplied by its area was used as signal intensity for further analysis.
|
| Description |
none
|
| Data processing |
Raw intensity data were imported into the R 2.9.0 software after preprocessing it with custom made Perl scripts. Further analysis was carried out using the LIMMA package of BIOCONDUCTOR. Background correction was done using the normexp method. Normalization of data within arrays was done using the ‘loess’ method. To normalize the data between arrays the ‘quantile’ method was used. The microarray data for each gene were fitted to a linear model, and statistics were generated using the lmFit and eBayes functions of the LIMMA package. The P values were adjusted for multiple testing using the Benjamini and Hochberg method. Genes with fold change ≥ 2 were considered as differentially expressed.
|
| |
|
| Submission date |
Aug 03, 2009 |
| Last update date |
Aug 03, 2009 |
| Contact name |
Endre Sebestyén |
| Organization name |
Semmelweis University
|
| Department |
1st Department of Pathology and Experimental Cancer Research
|
| Street address |
Üllői út 26.
|
| City |
Budapest |
| ZIP/Postal code |
1085 |
| Country |
Hungary |
| |
|
| Platform ID |
GPL6438 |
| Series (1) |
| GSE17484 |
Transcriptome analysis of germinating maize kernels exposed to smoke-water and the active compound KAR1 |
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