Berries of the cultivar Moscato Bianco (Vitis vinifera L.) were sampled in 2006 from pre-véraison to over-ripening, for a total of 13 collection dates. At each time point, ten bunches were taken from ten plants out of the ~250 grown in the experimental field “Inferno” of FEM (Fondazione Edmund Mach, San Michele all’Adige, Italy). Care was paid to sample from different vines and positions within each vine. In the lab, berries were pooled in order to minimize environmental effects. Berries were hand-peeled, the skins were immediately frozen in liquid nitrogen and stored at -80 °C until RNA extraction. Five of the 13 time points were used for microarray analysis.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted from grape skins using the SpectrumTM Plant Total RNA Kit (Sigma-Aldrich, St. Louis, Missouri, USA). RNA quantity and quality were evaluated with a NanoDrop ND-8000 spectrophotometer (NanoDrop Technologies, Wilmington, Delaware, USA) and an Agilent 2100 Bioanalyzer (Agilent Technologies, Mississauga, Ontario, Canada).
Label
Cy3
Label protocol
Fluorescently (Cy3/Cy5) labelled antisense RNA (aRNA) targets were obtained using the Amino Allyl MessageAmpTM II aRNA Amplification Kit (Ambion, Austin, Texas, USA) according to manufacturer’s instructions. Six hundred pmol Cy3- and Cy5-labelled aRNA were pooled together, fragmented and adjusted to 100 µL with hybridization buffer.
Berries of the cultivar Moscato Bianco (Vitis vinifera L.) were sampled in 2006 from pre-véraison to over-ripening, for a total of 13 collection dates. At each time point, ten bunches were taken from ten plants out of the ~250 grown in the experimental field “Inferno” of FEM (Fondazione Edmund Mach, San Michele all’Adige, Italy). Care was paid to sample from different vines and positions within each vine. In the lab, berries were pooled in order to minimize environmental effects. Berries were hand-peeled, the skins were immediately frozen in liquid nitrogen and stored at -80 °C until RNA extraction. Five of the 13 time points were used for microarray analysis.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted from grape skins using the SpectrumTM Plant Total RNA Kit (Sigma-Aldrich, St. Louis, Missouri, USA). RNA quantity and quality were evaluated with a NanoDrop ND-8000 spectrophotometer (NanoDrop Technologies, Wilmington, Delaware, USA) and an Agilent 2100 Bioanalyzer (Agilent Technologies, Mississauga, Ontario, Canada).
Label
Cy5
Label protocol
Fluorescently (Cy3/Cy5) labelled antisense RNA (aRNA) targets were obtained using the Amino Allyl MessageAmpTM II aRNA Amplification Kit (Ambion, Austin, Texas, USA) according to manufacturer’s instructions. Six hundred pmol Cy3- and Cy5-labelled aRNA were pooled together, fragmented and adjusted to 100 µL with hybridization buffer.
Hybridization protocol
Just before hybridization, the oligos were fixed on the chip by exposure to 100 MJ of UV light in a UV Stratalinker 2400 crosslinker (Stratagene, La Jolla, California, USA). The slides were then soaked twice in 0.2% sodium dodecyl sulfate (SDS) for 1 min. The probe solution was denatured at 100 °C for 1 min, cooled on ice for 2 min, stabilized at 37 °C for 5 min and injected into the hybridization chamber of the HS 4800 Hybridization Station (Tecan, Durham, North Carolina, USA). The slides were incubated at 37 °C for 16 h with medium agitation and then washed sequentially at 30 °C in 1X saline-sodium citrate (SSC)/0.1% SDS for 1 min 3 times, in 0.1X SSC/0.1% SDS for 1 min 3 times and finally in 0.1X SSC for 30 s.
Scan protocol
Washed slides were quickly dried with nitrogen and immediately scanned at 532 (Cy3) and 635 (Cy5) nm with GenePix 4000B fluorescence reader (Molecular Devices, Sunnyvale, California, USA) using GenePix 4.0 software. Parameters were fixed as follows: 400 V at 532 nm and 460 V at 635 nm for PTM (photomultiplier), 100% power, 5 for pixel size and 3 lines to average.
Description
Vitis vinifera L.
Data processing
Spot intensities were quantified with the software MAIA 2.75 (Novikov and Barillot, 2007). After excluding bad quality spots, median intensity gene expression data without background subtraction were normalized by a global lowess method followed by a print-tip median method with a modified version of the Goulphar script version 1.1.2 (Lemoine et al., 2006).
