tissue: phelloderm time of collection: am replicate: 3 slide batch: KB001 microarray scanner: PowerScanner (Tecan Group Ltd., Männedorf, Switzerland) image analysis program: ArrayPro Analyzer v. 6.3 (Media Cybernetics, Bethesda, MD, USA)
Growth protocol
Nursery planting stock (from open-pollinated seed lots) were obtained as 3-year-old seedlings of Picea glauca, Picea mariana, Picea abies, Picea Sitchensis, Pinus strobus, Pinus resinosa and Larix laricina, were transferred to 8-inch pots and grown in a greenhouse under natural light conditions. Sampling of tissues was timed with the ending of primary shoot elongation, i.e. after 6 to 8 weeks of growth, and was as described (Pavy et al. 2008). For each tissue type five biological samples were prepared by pooling 6 independent trees.
Extracted molecule
total RNA
Extraction protocol
All of the tissue samples were frozen in liquid nitrogen immediately after removal from the trees 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.
Label
Alexa 555
Label protocol
Total RNA (1 µg) 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
Slide scanning was performed at 5 micron resolution. Microarray scanner and image analysis software used are described in the Samples section
Description
KB001-14829
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). 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.
