|
| Status |
Public on Jan 30, 2012 |
| Title |
Collection day 0 biological replicate 3, technical replicate 2 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Day0_3_2
|
| Organism |
Pinus pinaster |
| Characteristics |
tissue: Zygotic embryos
stages: T0, T1 and T2
pool: 60 embryos
|
| Growth protocol |
Maritime pine immature female cones were randomly collected from open-pollinated plus trees growing in a clonal orchard at Escaroupim National Forest, Portugal. Cone collection was performed at five different dates, from 3-5 trees at each date, during July and August 2007. Seeds were removed from cones and opened for isolation of the dominant embryo. Embryos were quickly observed under a dissecting stereomicroscope for evaluation of developmental stage according to the staging system of Pullman and Webb (Pullman and Webb. TAPPI R&D Division Biological Sciences Symposium 1994) as described by Gonçalves et al. (Gonçalves et al. Planta 2005, 222(3):556–563). Embryos were pooled into five different developmental periods according to the collection date, with each collection date containing one or several consecutive embryo developmental stages as follows: Day 0 (stages T0, T1 and T2); Day 5 (stages T3 and T4); Day 11 (stage T4B); Day 15 (stage T5) and Day 25 (stage T7). Embryo samples were kept frozen at -80ºC until total RNA extraction.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA extracted using RNeasy Plant Mini Kit (Qiagen). On-column DNaseI digestion was performed with the RNase-Free DNase Set (Qiagen) according to manufacturer’s instructions. In some cases total RNA from different extractions from the same collection date was pooled to reach a total of 60 embryos in each sample.
|
| Label |
Cy3
|
| Label protocol |
Messenger RNA amplification and amino allyl UTP nucleotide incorporation were performed using Amino Allyl MessageAmp II aRNA Amplification Kit (Ambion) after treatment with TURBO DNase (Ambion). One hundred nanograms of DNase-treated total RNA was amplified in two sequential rounds of amplification following instructions provided by the supplier. Sample fluorescent labeling was performed according to Lorenz et al. (Lorenz et al. Journal of Visualized Experiments 2009, 25).
|
| |
|
| Channel 2 |
| Source name |
Reference sample
|
| Organism |
Pinus pinaster |
| Characteristics |
sample type: Reference sample composed by a pool of 200 ng of total RNA from each biological replicate of Day0, Day5, Day11, Day15 and Day25.
|
| Growth protocol |
Maritime pine immature female cones were randomly collected from open-pollinated plus trees growing in a clonal orchard at Escaroupim National Forest, Portugal. Cone collection was performed at five different dates, from 3-5 trees at each date, during July and August 2007. Seeds were removed from cones and opened for isolation of the dominant embryo. Embryos were quickly observed under a dissecting stereomicroscope for evaluation of developmental stage according to the staging system of Pullman and Webb (Pullman and Webb. TAPPI R&D Division Biological Sciences Symposium 1994) as described by Gonçalves et al. (Gonçalves et al. Planta 2005, 222(3):556–563). Embryos were pooled into five different developmental periods according to the collection date, with each collection date containing one or several consecutive embryo developmental stages as follows: Day 0 (stages T0, T1 and T2); Day 5 (stages T3 and T4); Day 11 (stage T4B); Day 15 (stage T5) and Day 25 (stage T7). Embryo samples were kept frozen at -80ºC until total RNA extraction.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA extracted using RNeasy Plant Mini Kit (Qiagen). On-column DNaseI digestion was performed with the RNase-Free DNase Set (Qiagen) according to manufacturer’s instructions. In some cases total RNA from different extractions from the same collection date was pooled to reach a total of 60 embryos in each sample.
|
| Label |
Cy5
|
| Label protocol |
Messenger RNA amplification and amino allyl UTP nucleotide incorporation were performed using Amino Allyl MessageAmp II aRNA Amplification Kit (Ambion) after treatment with TURBO DNase (Ambion). One hundred nanograms of DNase-treated total RNA was amplified in two sequential rounds of amplification following instructions provided by the supplier. Sample fluorescent labeling was performed according to Lorenz et al. (Lorenz et al. Journal of Visualized Experiments 2009, 25).
|
| |
|
| |
| Hybridization protocol |
Hybridization and pre and post-hybridization washing steps were performed according to Lorenz et al. (Lorenz et al. Journal of Visualized Experiments 2009, 25) except that 2 μg of Cy-labeled amplified RNA was used to hybridize with the array after fragmentation with Fragmentation Reagent (Ambion, Cat. #AM8740).
|
| Scan protocol |
Slides were scanned on a ProScanArray™ confocal scanner (Perkin Elmer) equipped with 532 nm and 635 nm lasers.
|
| Data processing |
Numerical signal data was obtained using ImaGene software v6.1 (Biodiscovery). Poor quality spots (i.e. merged, excessively large or small diameter spots) that passed the specified thresholds were flagged manually. Duplicated probes as well as empty spots that were on the array to serve as internal controls were also flagged. Raw microarray data was then filtered using the methods described by Wang et al. (Wang et al. Nucleic Acids Res 2001, 29:e75) and converted into a file adequate to be imported into BRB-Array Tools software v3.7 (http://linus.nci.nih.gov/BRB-ArrayTools.html) by using an in house Perl script. Raw signal intensity data from the 30 slides were Log2 transformed and normalized by the print-tip Lowess method on BRB. Wang plus ImaGene flagging was used to filter signal spots. Samples were clustered hierarchically using centered correlation and average linkage methods in order to check for hybridization reproducibility. Biological replicate 2 from Day15 (samples Day15_2_1 and Day15_2_2) was excluded from further analyses for not fullfil the clustering requirements. Other users are advised to do the same. To investigate differential expressed gene patterns along embryo development a time course analysis was conducted on BRB with a maximum significance level and false discovery rate (FDR) of 0.001. This plug-in applies a regression model to identify genes whose expression is varying over time, based on BRB normalized Log2 ratio.
|
| |
|
| Submission date |
Oct 03, 2011 |
| Last update date |
Jan 30, 2012 |
| Contact name |
Marta Simões |
| E-mail(s) |
martas@itqb.unl.pt
|
| Organization name |
IBET
|
| Lab |
Forest Biotech
|
| Street address |
Quinta do Marquês - EAN
|
| City |
Oeiras |
| ZIP/Postal code |
2781-901 |
| Country |
Portugal |
| |
|
| Platform ID |
GPL11184 |
| Series (1) |
| GSE32551 |
Stage-specific transcriptional changes during zygotic embryogenesis in maritime pine (Pinus pinaster Ait.) |
|
