We collected endosperm tissues by first dissecting out the embryos, which were examined to determine the developmental stage. Then, using a glass pipette with a fine tip, the endosperm was extracted and collected into pools based on whether embryo development was at the globular-shape, heart-shape or cotyledon stage. During this process, care was taken to avoid tissue from the integument, but limited contamination cannot be ruled out. Amongst different sections of the endosperm, our tissue collections were enriched with micropylar endosperm surrounding the zygote.
Growth protocol
B. napus L. cv DH12075 was grown in 4-liter pots containing Terra-lite Redi-earth mix (W. R. Grace and Co., Ajax, ON, Canada). A total of 10 g of time-release (100-day) 14-14-14-fertilizer pellets were applied to the surface of each pot at the time of seeding. Each pot was watered daily with 200 ml of nutrient solution (150 μg ml−1 N, 62.5 μg ml−1 P, and 125 μg ml−1 K) at the early growth stage, with 400 ml of the solution from 3 weeks after sowing, and with 200 ml of the solution during seed maturation. The plants were grown in a greenhouse with 20/16°C (d/n) temperature, 16/8 h photoperiod, and 250 μmol m−2 s−1 light intensity.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted from frozen tissues at each embryo developmental stage using RNeasy Plant Mini Kit (Qiagen, Mississauga, ON) and on-column DNase digestion with RNase-free DNase Set (Qiagen) was performed for each sample during RNA isolation according to the protocol provided by the manufacturer. The yield and RNA purity were determined using a Nanodrop ND-1000 Spectrophotometer, and the intactness of RNA was verified by gel electrophoresis.
Label
Cy3
Label protocol
Approximately 1 µg total RNA was treated with DNase I and reverse transcribed into aRNA with MessageTM II aRNA Amplification Kit (Ambion, Austin, TX, USA) according to the manufacturer’s protocol. The aRNA was selected as the template for fluorescent target preparation for microarray experiments. The quality of amplification was identified using agarose gel electrophoresis, and the aRNA yield was quantified using a spectrophotometer DU®7400 (Beckman, USA). An aliquot of 3 μg of aRNA was reverse transcribed using an oligo(dT)20 primer and 400 units of Superscript III reverse transcriptase (Invitrogen, Cat. No.18080-044) in the presence of 25 mM dATP, 25 mM dCTP, 25 mM dGTP, 15 mM aminoallyl-dUTP (Ambion, Cat. No. AM8439) and 10 mM dTTP. The single stranded cDNA was purified using YM30 filter column (Millipore, Billerica, MA) and labeled with CyDye post-labeling dye (Amersham, UK) for 1 hr.
We collected endosperm tissues by first dissecting out the embryos, which were examined to determine the developmental stage. Then, using a glass pipette with a fine tip, the endosperm was extracted and collected into pools based on whether embryo development was at the globular-shape, heart-shape or cotyledon stage. During this process, care was taken to avoid tissue from the integument, but limited contamination cannot be ruled out. Amongst different sections of the endosperm, our tissue collections were enriched with micropylar endosperm surrounding the zygote.
Growth protocol
B. napus L. cv DH12075 was grown in 4-liter pots containing Terra-lite Redi-earth mix (W. R. Grace and Co., Ajax, ON, Canada). A total of 10 g of time-release (100-day) 14-14-14-fertilizer pellets were applied to the surface of each pot at the time of seeding. Each pot was watered daily with 200 ml of nutrient solution (150 μg ml−1 N, 62.5 μg ml−1 P, and 125 μg ml−1 K) at the early growth stage, with 400 ml of the solution from 3 weeks after sowing, and with 200 ml of the solution during seed maturation. The plants were grown in a greenhouse with 20/16°C (d/n) temperature, 16/8 h photoperiod, and 250 μmol m−2 s−1 light intensity.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted from frozen tissues at each embryo developmental stage using RNeasy Plant Mini Kit (Qiagen, Mississauga, ON) and on-column DNase digestion with RNase-free DNase Set (Qiagen) was performed for each sample during RNA isolation according to the protocol provided by the manufacturer. The yield and RNA purity were determined using a Nanodrop ND-1000 Spectrophotometer, and the intactness of RNA was verified by gel electrophoresis.
Label
Cy5
Label protocol
Approximately 1 µg total RNA was treated with DNase I and reverse transcribed into aRNA with MessageTM II aRNA Amplification Kit (Ambion, Austin, TX, USA) according to the manufacturer’s protocol. The aRNA was selected as the template for fluorescent target preparation for microarray experiments. The quality of amplification was identified using agarose gel electrophoresis, and the aRNA yield was quantified using a spectrophotometer DU®7400 (Beckman, USA). An aliquot of 3 μg of aRNA was reverse transcribed using an oligo(dT)20 primer and 400 units of Superscript III reverse transcriptase (Invitrogen, Cat. No.18080-044) in the presence of 25 mM dATP, 25 mM dCTP, 25 mM dGTP, 15 mM aminoallyl-dUTP (Ambion, Cat. No. AM8439) and 10 mM dTTP. The single stranded cDNA was purified using YM30 filter column (Millipore, Billerica, MA) and labeled with CyDye post-labeling dye (Amersham, UK) for 1 hr.
Hybridization protocol
After a second purification as above, both Cy5- and Cy3-labeled cDNAs were pooled. The mixture was concentrated using a vacuum dryer and resuspended in 30 µl hybridization buffer. The pre-hybridization and hybridization procedures were performed as previously described (Huang et al, Plant Mol Biol, 2006, 62, 579-591).
Scan protocol
Hybridized arrays were scanned using a Genepix 4000B microarray scanner (Axon Instruments, CA, USA) at 5 µm resolution, 100% laser power, and different PMT values to obtain a similar green and red overall intensity. Raw spot fluorescence intensities were collected using GenePix Pro version 6.0 (Axon Instruments, CA, USA). A quality control filter was used to flag questionable spots on the array so they could be removed from analysis.
Description
Endosperm at the globular-shape embryo stage (6 days after flowering (DAF)) and heart-shape embryo stage (8 DAF).
Data processing
Before normalization, a basic pre-processing was performed. The outlier spots with median intensity larger than that of (median ± 10×SD) were removed. Channel intensity was considered low if its background-corrected intensity value was less than a preset low-intensity threshold (LIT) of 0.5; low channel intensities were replaced by the LIT. The spot was marked as bad if either of the two channels was low. Spots with background-subtracted median intensity greater than median background intensity in at least one channel were selected and used for normalization and further analysis. Array features annotated as “DMSO,” “Blank” or “Empty” were flagged and excluded from analysis. A spot was removed if the fold change of the Cy3/Cy5 group was in the opposite direction of that of the Cy5/Cy3 group. For example, if spot A was up-regulated for the Cy3/CY5 group and down-regulated for the Cy5/Cy3 group, then spot A was removed because of its conflicting data. Foreground intensities of selected genes were background corrected using the normexp background measure, and the signal intensities were normalized using ‘global loess’ for within-array normalization and quantile for between-array normalization using R-package Limma. The signal intensity (background-corrected) of each spot on the microarray was combined by averaging two biological duplicates using Excel 2003 (Microsoft). After the normalization process, the signal intensities and log2 ratio of the two channels of each replicate were obtained. These two sets of data were processed through rank product implemented in the BioMiner software suite with a 5% false discovery rate. Genes with very inconsistent signal intensities and log2 ratio were excluded from this process. Then we combined the results from all three stages. The genes that ranked high in both datasets were considered differentially expressed genes and classified into patterns based on the clustering approach. The DNA sequence for each of the unisequences spotted on the array was searched for in the TAIR protein database (version 7) using BLASTX programs at an E value less than 10-5. The best match was extracted using an in-house Perl script and used as a basis for obtaining annotations for each probe based on sequence identity. The best matches were compared to terms of the Gene Ontology (GO) Consortium (GO). Categories were assigned based on biological, functional, and molecular annotations available from GO.
