The seed materials were obtained from Canadian Forest Service (CFS) seed bank in Quebec City, Canada (Dr J. Beaulieu). These seed lots were from a single tree and each seed lot was collected in a single year. The mother trees used in this study (77111 and 80112) correspond to trees studied by Pelgas et al. (BMC Genomics 12, 145 [2011]). Before stratification, the seeds were surface sterilized with 70% EtOH (1 min) and 3% Na-hypochlorite (10 min), rinsing the seeds three times with sterilized water between and after treatments. The seeds were stratified by immersing them in cold (4° Celsius) sterile water for 24 hours, followed by storage in 4° Celsius for 28 days. The germination process was then started by incubating the stratified seeds in 26° Celsius in the dark for four hours.
Extracted molecule
polyA RNA
Extraction protocol
The megagametophytes were dissected from the rest of the seeds under a dissecting microscope, separated in two halves of equal size, flash frozen on liquid nitrogen and store at -80 Celsius until used for RNA extraction. Megametophyte tissues were ground using a pestle in liquid nitrogen and mRNA was isolated using the Dynabeads mRNA Mikro kit (Dynal/Invitrogen, Oslo, Norway) following manufacturers instructions.The mRNA quality and concentration were determined with mRNA Pico assays (Agilent Technologies Inc., Santa Clara, CA, USA) on Agilent BioAnalyzer.
Label
Alexa555
Label protocol
The mRNA (9 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 and 647 dyes (Invitrogen, Carlsbad, CA, USA), one for each replicated sample set, 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 two random samples labelled with different dyes. The samples to be hybridized to a microarray 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
Slide scanning was performed with the PowerScanner (Tecan) microarray scanner at 5 micron resolution and image analysis was carried out using ArrayPro Analyzer v. 6.3 (Media Cybernetics, Bethesda, MD, USA).
Description
FLAG column defines whether spot had normal (1) or abnormal (0) morphology.
Data processing
The data analyses were carried out on data (trimmed spot intensity means) from the two channels separately. An RGList object was built for each replicate sample set (i.e. analysis channel) with Limma (G. K. Smyth, Bioinformatics and Computational Biology Solutions using R and Bioconductor, Springer [New York] [2005]) in the R statistical environment (R. Ihaka & R. Gentleman, J Comp Graph Stat 5, 299 [1996]). These single color microarray fluorescence data were then background-corrected and normalized using the normexp and quantile -methods.
Submission date
Jan 25, 2012
Last update date
Dec 01, 2012
Contact name
Jukka-Pekka Verta
Organization name
Centre for Forest Research
Department
Department of Wood and Forest Sciences
Lab
John J. MacKay
Street address
1030 Avenue de la Medecine, Pavillon Charles-Eugene-Marchand, bureau 2225
