normalization data transformation protocol
Spots selected for analysis were those that showed median intensity values higher than the background plus two-standard deviations of the background median in at least one channel. To generate raw data to be used for expression analysis all hybridization files were combined. Normalized Lowess M Log Ratio was used as expression value and patterns with more than 80% of missing values were filtered.
array scanning protocol
Slides were scanned with a GenePix 4000B scanner (Axon Instruments) at 10 um resolution, 100% laser power, and different PMT values to adjust the ratio to 1.0 and to cover all dynamic range of image. Microarray images were analyzed using GenePix 4.1 (Axon Instruments) software. (Parameters: Scanning hardware = Axon- GenePix4000B, Scanning software = GenePix Pro [Axon Instruments])
nucleic acid hybridization to array protocol
Microarray hybridization was performed manually on the ChillPeach microarray slides using Telechem Hybridization Chambers (Corning) and following manufacturer instructions. -Slides were re-hydrated and UV-cross-linked, - Slides were pre-hybridized 45 min at 42C in 5× SSC, 0.1%SDS, 0.1 mg/ml BSA, 10 mM EDTA pH 8, washed twice for 30 s in milliQ water (Millipore) and in isopropanol for 30 s. Arrays were drained by centrifugation at 528g for 2 min. For each hybridization, 100 pmol of each Cy5-labelled sample was mixed with 100 pmol of Cy3-labelled reference pool. Three replicates were made, one of them dye-swapped. Mix was dried in a speed-vac, and re-suspended in 34 ul water, 4 ul EDTA 0.5 M pH 8 and 2 ul polyA (10 ug/ul). This mix was denatured for 3 min at 95C and 40 ul of 2× hybridization buffer (50 ul formamide 50 ul, 25 ul 20× SSC, 2 ul 10%SDS) was added. Hybridization was done overnight at 42C. After hybridization, slides were washed in 2× SSC, 0.1% SDS for 5 min at 42C, 0.1× SSC, 0.1% SDS for 10 min at room temperature, 0.1× SSC for 5 min at room temperature four times, and 0.01× SSC for 5 min at room temperature four times. Arrays were drained by centrifugation at 528g for 2 min (Parameters: Chamber type = OTHER: elechem Hybridization Chambers (Corning), Quantity of label target used = 1, Mass unit = Micro gram, time = 16, Tiny time unit = hours, Volume = 80, Volume unit = Micro litre, temperature = 42)
nucleic acid labeling protocol
RNA for microarray hybridization was amplified using the method of Van Gelder et al. (1990). Briefly, 1 ?g of total RNA of each sample or superpool was amplified and aminoallyl-labelled using MessageAmp II aRNA kit (Ambion, http://www.ambion.com) and 5-(3-aminoallyl)-2'-deoxyuridine-5'-triphosphate (aa-dUTP, Ambion), following manufacturer’s instructions. Approximately 40–50 ug of amplified RNA (aRNA) was normally obtained. For each sample 7.5 ug of aminoallyl-labelled aRNA was re-suspended in 0.1 M Na2CO3 (pH 9.0) and labeled with either Cy3 or Cy5 Mono NHS Ester (CyTM Dye Postlabelling Reactive Dye Pack, Amersham). The samples were purified with MegaclearTM (Ambion) following manufacturer instructions. Incorporation of Cy3 and Cy5 was measured using 1 ul of the probe in a Nanodrop spectrophotometer (Nanodrop Technologies Inc.; http://www.nanodrop.com/). (Parameters: Amount of nucleic acid labeled = 1, Amplification = RNA polymerases, Mass unit = Micro gram)
Treatments are conducted in both sensitive and tolerant genotypes: Mature (M-samples): fruits harvested at the mature stage corresponding to 12–14 lb fruit firmness. Ripening (R-samples): M fruits were let to ripen R at 20C to 2–3 lb firmness. MI induction (CS-samples): M fruits from the different genotypes were forced-air cooled at 0–2C within 6 h of harvest and then stored at 5C with 90% relative humidity for 1, 2 and 3 weeks (M+cold) .
For each treatment (M,R , CS, CSR ) and genotype (S and T) there are created pools using: M Sensitive pool: equal amounts of RNA samples from 49/59, 84/85, 86/87 and 132/133 Pop-DG lines in M stage. R Sensitive pool: equal amounts of RNA samples from 49/59, 84/85, 86/87 and 132/133 Pop-DG lines from R samples. CS Sensitive pools: equal amounts of RNA samples from 49/59, 84/85, 86/87 and 132/133 Pop-DG lines stored 1, 2 or 3weeks in cold. CSR Sensitive pool: equal amounts of RNA samples from 49/59, 84/85, 86/87 and 132/133 Pop-DG lines stored 1, 2 or 3weeks in cold and shelf life ripened. M-Tolerant pool: equal amounts of RNA samples from 71/72, 88/89, 134/135, 142/143 PopDG lines in M stage. R-Tolerant pool: equal amounts of RNA samples from 71/72, 88/89, 134/135, 142/143 PopDG lines from R samples. CS-Tolerant pools: equal amounts of RNA samples from 71/72, 88/89, 134/135, 142/143 PopDG lines stored 1, 2 or 3weeks in cold. CSR-Tolerant pools: equal amounts of RNA samples from 71/72, 88/89, 134/135, 142/143 PopDG lines stored 1, 2 or 3weeks in cold and shelf life ripened. Reference pool or superpool: equal amounts of RNA samples from 49/59, 84/85, 86/87, 132/133, 71/72, 88/89, 134/135, 142/143 PopDG lines of M, R, CS and CSR treatments
nucleic acid extraction protocol
Total RNA was isolated using the method described by Meisel et al. (2005) from 4 g of mesocarp tissue. (Parameters: Extracted product = total_RNA, Amplification = none)