tissue: megagametophytes age of trees: 0 genotype: 80112 replicate: 1 slide batch: JF003
Growth protocol
Control-pollinated seed (cross C962856) were obtained from P. glauca tree (80112) described in Pelgas et al. (2011) were surface-sterilized for 1 minute in 70% EtOH and ten minutes in 3% Na-hypochlorite, washing the seeds with sterile water three times in after and between treatments. The seeds were then immersed at 4°C sterile water for 24 hours and stratified at 4°C for 28 days. Next, the seeds were, moved to 26 Celsius on petri dishes with a moist paper and kept in the dark °C for 16h to start the germination process. After four hours of incubation the seeds were opened and the embryo was separated from the megagametophyte under a dissecting microscope. A total of 3 biological samples comprised of a single megagametophyte were used for analysis.
Extracted molecule
polyA RNA
Extraction protocol
All of the tissue samples were frozen in liquid nitrogen immediately after removal from the trees, the seed or tissue culture vessels, and stored at -80°C. Tissues were ground under liquid nitrogen and total RNA was extracted following Chang et al. (1993) as described in Pavy et al. (2008) for all of the sample types, except for megagametophytes where poly A+ RNA was extracted directly by using the Dynabeads mRNA Mikro kit (Dynal/Invitrogen, Oslo, Norway) following manufacturers instructions.
Label
Alexa647
Label protocol
Total RNA (1 µg) or mRNA (10 ng) was transcribed in-vitro by using the Amino Allyl MessageAmp II aRNA Amplification kit (Ambion by Life Technologies, Austin, TX, USA), following the manufacturer’s instructions. The aaRNA (5 ug per sample) was labeled using Alexa Fluor 555 or 647 dyes (Invitrogen, Carlsbad, CA, USA), and purified as per the manufacturer’s instructions. Dye incorporation efficiency was determined by using a Nanodrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturer’s instructions.
Hybridization protocol
Each microarray was hybridized with one sample labelled with one dye. The samples were mixed and the volume was reduced to ~10 µl by evaporating excess water in a DNA 120 speedvac (Thermo Fisher Scientific). Labelled aRNAs were fragmented for 15 minutes at 70°C using Ambion’s ”RNA Fragmentation Reagents“, placed on ice for 1 minute, denatured for 2 minutes at 95°C, put on ice for 2 min and resuspended in 120 µl hybridization buffer (50% formamide, 5X SSC, 0,1% SDS, 0,1 mg/mL Herring sperm DNA) preheated to 55°C. Samples were kept in a heating block at 50°C until hybridization. Hybridizations were performed in HS400Pro hybridization stations (Tecan Group Ltd., Männedorf, Switzerland). The slides were heated at 80°C for 10 minutes, then washed once at 37°C with 0.5X SSC, 0.1% SDS for 20 seconds and once at 50°C with 2X SSC, 0.5% SDS for 20 seconds, and prehybridized for 1 hour at 65°C in 5X SSC, 0.1% SDS, 0.1 mg/ml BSA, 0.1 mg/ml Herring Sperm DNA. Next the slides were washed at 55°C with 2X SSC, 0.5% SDS for 1 minute with a 30 seconds soak and washed again at 45°C for 1 minute with the same solution. The resuspended labelled targets were injected into the chambers and hybridized for 16 hours at 45°C with sample agitation. The slides were then washed as follows: 2 times 1 minute 30 seconds at 45°C with 30 seconds soaking in 2X SSC, 0.5% SDS, 1 time 1 minute at 45° in 2X SSC, 0.5% SDS, 2 times 1 minute 30 seconds at 45°C with 30 seconds soaking in 0.5X SSC, 0.1% SDS, 1 time 1 minute at 37°C with 20 seconds soaking in 0.5X SSC, 0.1% SDS, 1 time 1 minute at 23°C with 20 seconds soaking in 0.5X SSC, 0.1% SDS, 1 time 1 minute 30 seconds at 23°C with 30 seconds soaking in 0.1X SSC, 1 time 30 seconds at 23°C in 0.1X SSC and 2 times 30 seconds at 23°C in milliQ filtered water. Finally slides were dried for 2 minutes 30 seconds with nitrogen gaz.
Scan protocol
Microarray Scanner: PowerScanner (Tecan Group Ltd., Männedorf, Switzerland) Image Analysis Program: ArrayPro Analyzer v. 6.3 (Media Cybernetics, Bethesda, MD, USA) Slide scanning was performed at 5 micron resolution.
Description
JF003-000000008138-635-2011-03-04.txt Sample label Alexa647 is accurate. However, the raw data file contains '532'-labeled columns because the image processing software labels the ch1 columns as '532' by default, whatever the actual dye used.
Data processing
Data analyses were performed using customized scripts for R and Bioconductor (http://www.r-project.org and http://www.bioconductor.org). Spots that were flagged as presenting abnormal morphology during the image processing were replaced by mean value of the remaining spots of the same probe from the other slides from the same sample type. Background intensities were subtracted from the foreground intensities. Background-subtracted data were log2-transformed and normalized using quantile correction approach. A filtering step was applied to select positive genes to be used for further analysis. The mean intensity of spots containing buffer only was calculated for each row of sub-grids, and was taken as the minimum intensity of probes for that subgrid. A probe was called positive (detected above background) when its signal intensity was above the buffer intensity on at least 50% of slides within a given sample type. When determining differential expression, positive probes were probes that were detected according to this criterion in at least one of the tissues (e.g. phelloderm and xylem juvenile). Mean probe intensity was determined for genes represented by more than one positive probe. The average signal intensity was determined from all of the slides available for a sample type. The genes were then ranked based on their average signal intensities within a tissue type and equally divided into 10 separate classes according to their signal intensity. Genes from class 1 or class 10 were the 10% with lowest and highest signal intensities, respectively.
