age: about 60 years tissue: five pooled shade leaves tree: 399 exposition: 1x ambient ozone
Extracted molecule
total RNA
Extraction protocol
According to Kiefer et al. 2000.
Label
Cy5
Label protocol
Cy5 Mono-Reactive Dye Pack dissolved in 85 µl DMSO, dried cDNA dissolved in 4.5µl Na2CO3, 4.5µl dissolved Cy5 dye was added and incubated for about 2 hours at room temperature and in the dark. Precipitate with 35 µl 100 mM NaAc (pH 5,2), purified with QIAquick PCR Purification Kit, Qiagen, Hilden. Dried and dissolved in 62 µl hybridization buffer (50% formamide, 5-fach SSC, 0.1% SDS, 2% salmon sperm DNA (w/w), then for 3 minutes at 90°C, centrifuged and put on array.
age: about 60 years tissue: five pooled shade leaves tree: 437 exposition: 2x ambient ozone
Extracted molecule
total RNA
Extraction protocol
According to Kiefer et al. 2000.
Label
Cy3
Label protocol
Cy3 Mono-Reactive Dye Pack dissolved in 85 µl DMSO, dried cDNA dissolved in 4.5µl Na2CO3, 4.5µl dissolved Cy3 dye was added and incubated for about 2 hours at room temperature and in the dark. Precipitate with 35 µl 100 mM NaAc (pH 5,2), purified with QIAquick PCR Purification Kit, Qiagen, Hilden. Dried and dissolved in 62 µl hybridization buffer (50% formamide, 5-fach SSC, 0.1% SDS, 2% salmon sperm DNA (w/w), then for 3 minutes at 90°C, centrifuged and put on array.
Hybridization protocol
Blocking: 1. 0.1 % SDS for 2 minutes shaking, 2. 0.1 % SDS for 2 minutes shaking, 3. H2Obidest. for 2 minutes shaking, 4. washing of slides in H2Obidest., 5. washing another time with water for 2 minutes, 6. for 2 minutes in cooking water, 7. for 5 minutes in fresh sodium-boric-solution (0.75 g NaBH4 in 200 ml PBS and 75 ml 100% Ethanol p.a.), 8. 1 minutes washing in 0.1 % SDS, 9. another time washing in 0.1 % SDS for 1 minute, 10. 1 minute washing in H2Obidest, 11. incubating in 100% EtOH for about 20 seconds. Hybridization for 16h up to 20h at 42°C in a hybridization furnace. Washing: in wash buffer (1x SSC/0.2% SDS) for 4 minutes at 42 °C, 4 minutes in wash buffer (0.1x SSC und 0.2% SDS), 2 minutes in wash buffer (0.1x SSC), 2 minutes in wash buffer (0.1x SSC), 2 minutes in Millipore water.
Scan protocol
Hardware: GenePix 4000A (Axon Instruments, Sunnyvale, USA), simultaneous scanning of both wavelengths (532nm, 635nm). Software: GenePix Pro 6.0 (Axon Instruments (Sunnyvale, USA) FLA 8000 Version 1.3.
Description
A coefficient of variation about all microarrays of one time point was calculated using Acuity 4.0 microarray informatics software [Axon Instruments]. We used only those spots that had a coefficient of variation < 50 and were present on at least half of identical slides. We express the median values as log2 ratios.
Data processing
Normalization via GenePix Pro 4.0. For normalization, only spots were used which (1) were not marked with "bad", "absent", or "not found"; (2) spots that had for both wavelengths more than 55% of pixel above background one standard deviation; (3) spots with a pixel intensity for both wavelengths above 500; (4) coefficient of determination of actual regression of both wavelengths was above 0.5; (5) light saturation of pixel was under 3%. On the basis of these values, all genes of the array were normalized.
