7 protocols
normalization data transformation protocol
The statistical processing of the data was conducted separately on the two lines and different leaf samples by using identical procedures. The low quality spots were downweighted; these included spots with the sum of median pixel intensities less than 300 and spots having the feature pixel intensity less than 55 % above one standard deviation of the background pixel intensity in both wavelengths. The data was normalized by using print-tip loess within and Aquantile (Aq) between array normalizations. ID_REF = VALUE = Print-tip loess and Aq-normalized log2 ratios (Cy5/Cy3) calculated using limma
array scanning protocol
Slides were scanned at 633 nm and 543 nm at 98 % laser power, at a resolution of 10 µm with varying PMT values with ScanArray® Gx PLUS (Microarray Scanner, PerkinElmer, Waltham, MA, USA). The fixed-circle method was used in the quantification of spots, and the total-algorithm in the normalization was conducted with the ScanArray Express software (PerkinElmer).
hybridization protocol
Hybridizations and post-washings were conducted following the instructions of 3DNA Array 50 Expression Array Detection Kit (Genisphere Inc).
labelling protocol
The SuperScript III reverse transcriptase (Invitrogen, Carlsbad, CA, USA) was used to synthesize cDNAs in accordance with the manufacturer's instructions and the instructions of the 3DNA Array 50 Expression Array Detection Kit For Microarrays (Genisphere Inc, Hatfield, PA, USA).
sample treatment protocol
The saplings of wt and VHb lines grown 1.5 months under standard greenhouse conditions were used in the experiments. In the herbivory treatment, one larvae of Conistra vaccinii L. (Lepidoptera: Noctuidae) was placed on the leaf inside the veil cloth bag for 24 hours, which after samples were collected to liquid nitrogen.
nucleic acid extraction protocol
The total RNA was extracted as described by Jaakola et al. (Mol. Biotechnol. 19 (2001) 201-203) except for the first centrifugation step which was omitted.
growth protocol
The lines were multiplicated on liquid MS medium (full strength of C10H12FeN2NaO8; half strength of other micro and macro nutrients; 2.22 μM BA and 2.85 μM IAA) in RITA® temporary immersion containers (Vitropic, Saint-Mathieu-de-Tréviers, France) under 16:8 h light/dark photoperiod (110-130 μmol m-2s-1) at 22 ºC. Saplings were rooted on MS without plant growth regulators, acclimated in the Botanical Gardens of the University of Oulu (65°03'N, 25°27'E), and potted to limed and fertilised soil, and grown under standard greenhouse conditions.