|
| Status |
Public on Dec 03, 2015 |
| Title |
pCRE1_XVE_MIR165A_0h_rep2 |
| Sample type |
RNA |
| |
|
| Source name |
Arabidopsis thaliana root tips (0.5mm) before MIR165 induction
|
| Organism |
Arabidopsis thaliana |
| Characteristics |
tissue: 0.5 mm of primary root tip, 4 dpg
|
| Treatment protocol |
For transgenic inductions, seedlings were transferred to plates containing 5 µM ß-estradiol in ethanol, or a mock treatment containing the same amount of only ethanol for the indicated time, either 0h (mock plates), 6h, 10h, or 24h and seedlings were collected at 4 days post-germination. Each time point and genotype was collected in triplicate. Inductions and collections were staggered such that seedlings were the same age (+/- 4.5h) when collected.
|
| Growth protocol |
Plants were grown on square plates with 25 µm pore Sefar mesh placed on top of growth media (0.5X Murashige and Skoog (MS) medium, 1% sucrose, 1% plant agar). Plants were grown veritcally in a Sanyo MLR growth cabinet at 22ºC with 18h light and 6h dark (145-150 µmol m-2 s-1).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Root tips (0.5 mm) were hand-dissected from 4 dpg seedling primary roots and collected in RLT buffer from the Plant RNeasy kit (QIAGEN) containing ß-mercaptoethanol according to kit instructions. About 200 root tips were collected per replicate. Tissue was immediately homogenized with glass beads using a FastPrep homogenizer (Savant), and RNA extraction including the on-column RNase-free DNase digestion (QIAGEN) were performed following the manufacturer’s protocol.
|
| Label |
biotin
|
| Label protocol |
250 ng of total RNA from each sample were used to prepare biotinylated fragmented cRNA according to the GeneChip® 3’ IVT Express Kit Manual.
|
| |
|
| Hybridization protocol |
Affymetrix Arabidopsis ATH1 GeneChip® expression arrays were hybridized for 16h in a 45 °C incubator, rotated at 60 rpm. According to the GeneChip® Expression Wash, Stain and Scan Manual (PN 702731 Rev2, Affymetrix Inc., Santa Clara, CA) the arrays were then washed and stained using the Fluidics Station 450.
|
| Scan protocol |
The arrays were scanned using the GeneChip® Scanner 3000 7G.
|
| Description |
gene expression data of non-induced root tips in Col-0 background
|
| Data processing |
The raw data was normalized in the free software Expression Console provided by Affymetrix (http://www.affymetrix.com) using the robust multi-array average (RMA) method (Li and Wong, 2001; Irizarry et al., 2003). Subsequent analysis of the gene expression data was carried out in R (http://www.r-project.org) using Bioconductor packages (www.bioconductor.org). To search for differentially expressed genes between the different biological replicate groups within each experiment, an empirical Bayes moderated t-test was then applied (Smyth, 2004) using the ‘limma’ package (Smyth et al., 2005). To address the problem with multiple testing, the p-values were adjusted using the method of (Benjamini and Hochberg, 1995).
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| |
|
| Submission date |
Dec 02, 2015 |
| Last update date |
Dec 03, 2015 |
| Contact name |
Christina Joy Müller |
| Organization name |
Swedish University of Agricultural Sciences
|
| Department |
Plant Biology
|
| Street address |
Almas Allé 5
|
| City |
Uppsala |
| ZIP/Postal code |
75651 |
| Country |
Sweden |
| |
|
| Platform ID |
GPL198 |
| Series (2) |
| GSE75609 |
Gene expression in 0.5 mm root tips of Arabidopsis upon induction of pCRE1>>MIR165A |
| GSE75612 |
Gene expression in 0.5 mm root tips of Arabidopsis |
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