tissue: needles slide batch: KB001 resistance to budworm: Resistant position of sample in tree: Left tree number: 6
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 using a Mixer Mill MM 300 (Retsch, Haan, Germany) and total RNA was extracted following Chang et al. (1993) as described in Pavy et al. (2008).
Label
Alexa 647
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 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 on a PowerScanner (Tecan Group Ltd., Männedorf, Switzerland) microarray scanner at 50% laser power with the autogain funtion on to automatically adjust PMT level. Image analysis was carried out using ArrayPro Analyzer v. 6.3 (Media Cybernetics, Bethesda, MD, USA).
Data processing
Mean intensity and local background were obtained for each spot on the array. Bad spots resulting from dust, high local background or saturation were flagged. Net intensity was calculated by subtracting trimmed background from mean intensity. Data analysis was carried out using the FlexArray 1.6 software package (http://genomequebec.mcgill.ca/FlexArray). 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-subtracted data were log2-transformed and quantile normalization was used to normalize intensity distributions between arrays.
