tissue: adventitious roots age of trees: 1 genotype: 20
Growth protocol
Norway spruce (P. abies) seedlings were grown in the experimental nursery of Finnish Forest Research Institute for about one and a half years before sampling. They were grown in standard nursery growing media, light Sphagnum peat, and fertilized with mineral nutrients. Roots were washed under tap water to remove surrounding peat and approximately 1 cm of the root ends was collected for analysis. Between two and four biological samples comprised of several root ends were analysed for each of eight different genotypes. Sampling of Norway spruce cutting roots representing eight genotypes was done in late September; for 1st year cuttings five months and the 2nd year cuttings 18 months after adventitious root initiation. The adventitious roots were washed carefully under tap water and the short roots were immediately detached from the cutting, wrapped in aluminium foil and finally frozen in liquid nitrogen. Samples were stored at -70°C degrees until RNA extraction.
Extracted molecule
polyA RNA
Extraction protocol
Total RNA was extracted from five replicates per clone per sampling point (5replicates x 8 clones x 2 time points) in a random order. The RNA was extracted by grinding the adventitious roots in liquid nitrogen from 62 samples as described in Chang et al. (1993) with modifications as described in Pavy et al. (2008). Quantification and quality check of messenger RNA (mRNA) was done on a Nanodrop 1000 (Thermo Scientific, MA, US) spectrophotometer and the integrity was verified on the Bioanalyzer 2100 (Agilent Technologies, CA, US).
Label
AlexaFluor 647
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 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.
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. Some genes on the array (1017 pcs.) were represented by more than one probe. The gene intensity values of redundant probes were corrected replacing the raw values with the geometric mean of corresponding probe intensities (analysis done based on the cluster-gene names) so that each gene was finally represented only once in the analyses. The slides were categorized by the age of saplings into two sets (five and 18-month-old), and a probe was considered positive if the signal intensity was detected above buffer intensity on at least three quarters of the slides in either of the sampling points.
