One lot of fruit was allowed to ripen at room temperature (25°C) and fruit mesocarp (wedged-shape slices) was removed after 1, 3 and 5 days, respectively. The other lots were subjected to cold storage (0°C, R.H. 95%) for 4 and 6 weeks and mesocarp was sampled after removal from cold storage plus 1 day at room temperature. For each of the examined cultivars a common reference design was followed by using 0 weeks of cold storage plus 1 day of shelf life as a reference. Comparisons were carried out between (a) after harvest + 1 day of shelf life vs 4 weeks of cold storage + 1 day of shelf life and (b) after harvest + 1 day of shelf life vs 6 weeks + 1 day of shelf life. At least three biological replicates were created, repeating three times the same combination of targets of which one was a dye swap (in order to increase accuracy and to correct for dye-related differences).
Growth protocol
Fruit material from the cultivars ‘Royal Glory’ and ‘Morettini No 2’ was used in the current study. ‘Royal Glory’ fruit are characterized by high tissue firmness retention that can fully ripen on the tree while still retaining satisfactory firmness and acceptable quality attributes after harvest or after removal from cold storage, without incidence of CI symptoms, while ‘Morettini No 2’ fruit are characterized by low tissue firmness and high susceptibility to physiological disorders (internal breakdown and flesh wooliness) after extended cold storage (Manganaris et al., 2008a). Fruit of both cultivars were collected during the last developmental stages, corresponding to 70, 77 and 84 days after full bloom (DAFB), respectively. These stages correspond to 4, 3 and 2 weeks before harvest, respectively. Fruit harvested at commercially mature stage, according to fruit size and skin background colour, were separated into three 30-fruit lots.
Extracted molecule
total RNA
Extraction protocol
Frozen fruit (3 g) was ground in liquid nitrogen to a fine powder and total RNA was extracted as described by Ruperti and co-workers (Physiologia. Plantarum 111, 336-344). Fifty μg of total RNA were treated with 10 units of RQ1 RNase-Free DNAse (Promega) and 1 unit of RNAguard (RNase INHIBITOR) (Amersham) for 30 min, and then purified by phenol-chlorophorm according to the manufacturer’s instructions. The concentration of RNA was quantified by measuring the absorbance at 260 nm and its integrity was checked electrophoretically on agarose gels.
Label
Cy5,Cy3
Label protocol
Total RNA (20 μg) was converted into target cDNA by reverse transcription using the SuperScriptTM Indirect cDNA Labeling System (Invitrogen, USA) following manufacturer instruction. The amino-modified cDNA was coupled to a monoreactive N-hydroxysuccinimide (NHS)-ester fluorescent dyes: the red-fluorescent cyanine5 (Cy5) and the green-fluorescent cyanine3 (Cy3) (GE Healthcare, USA). A final purification step using Microcon-PCR centrifugal filter devices (Millipore, USA) removed any unincorporated dye. The purity and yield of the labelled cDNA was calculated from the OD values obtained by means of a spectrophotometer using the formulas reported in the SuperScriptTM Indirect cDNA Labeling System instruction manual.
Channel 2
Source name
fruit stored for 0 weeks at 0° plus 1 day at room temperature
One lot of fruit was allowed to ripen at room temperature (25°C) and fruit mesocarp (wedged-shape slices) was removed after 1, 3 and 5 days, respectively. The other lots were subjected to cold storage (0°C, R.H. 95%) for 4 and 6 weeks and mesocarp was sampled after removal from cold storage plus 1 day at room temperature. For each of the examined cultivars a common reference design was followed by using 0 weeks of cold storage plus 1 day of shelf life as a reference. Comparisons were carried out between (a) after harvest + 1 day of shelf life vs 4 weeks of cold storage + 1 day of shelf life and (b) after harvest + 1 day of shelf life vs 6 weeks + 1 day of shelf life. At least three biological replicates were created, repeating three times the same combination of targets of which one was a dye swap (in order to increase accuracy and to correct for dye-related differences).
Growth protocol
Fruit material from the cultivars ‘Royal Glory’ and ‘Morettini No 2’ was used in the current study. ‘Royal Glory’ fruit are characterized by high tissue firmness retention that can fully ripen on the tree while still retaining satisfactory firmness and acceptable quality attributes after harvest or after removal from cold storage, without incidence of CI symptoms, while ‘Morettini No 2’ fruit are characterized by low tissue firmness and high susceptibility to physiological disorders (internal breakdown and flesh wooliness) after extended cold storage (Manganaris et al., 2008a). Fruit of both cultivars were collected during the last developmental stages, corresponding to 70, 77 and 84 days after full bloom (DAFB), respectively. These stages correspond to 4, 3 and 2 weeks before harvest, respectively. Fruit harvested at commercially mature stage, according to fruit size and skin background colour, were separated into three 30-fruit lots.
Extracted molecule
total RNA
Extraction protocol
Frozen fruit (3 g) was ground in liquid nitrogen to a fine powder and total RNA was extracted as described by Ruperti and co-workers (Physiologia. Plantarum 111, 336-344). Fifty μg of total RNA were treated with 10 units of RQ1 RNase-Free DNAse (Promega) and 1 unit of RNAguard (RNase INHIBITOR) (Amersham) for 30 min, and then purified by phenol-chlorophorm according to the manufacturer’s instructions. The concentration of RNA was quantified by measuring the absorbance at 260 nm and its integrity was checked electrophoretically on agarose gels.
Label
Cy5,Cy3
Label protocol
Total RNA (20 μg) was converted into target cDNA by reverse transcription using the SuperScriptTM Indirect cDNA Labeling System (Invitrogen, USA) following manufacturer instruction. The amino-modified cDNA was coupled to a monoreactive N-hydroxysuccinimide (NHS)-ester fluorescent dyes: the red-fluorescent cyanine5 (Cy5) and the green-fluorescent cyanine3 (Cy3) (GE Healthcare, USA). A final purification step using Microcon-PCR centrifugal filter devices (Millipore, USA) removed any unincorporated dye. The purity and yield of the labelled cDNA was calculated from the OD values obtained by means of a spectrophotometer using the formulas reported in the SuperScriptTM Indirect cDNA Labeling System instruction manual.
Hybridization protocol
Pre-hybridisations were carried out by soaking whole glass slides in a solution containing 5X SSC, 0.1% SDS, 5X Denhardt’s solution and 100ng/μL DNA carrier at 48°C for at least 2 h. Then the slides were washed once with a 0.2X SSC solution and dried by centrifuging for 4 min at 1000 rpm. Hybridisations were carried out in 250 µL of hybridisation solution (5X SSC, 0.1% SDS, 25% formamide) containing 90-100 pmol of Cy3- and Cy5-labelled target cDNAs. The hybridisation solution was kept in place by means of a microarray gene frame (ABgene, UK), while the glass slides were placed in a hybridisation chamber (HybChamber, by Genomic Solutions, USA) kept on a rotary oven for at least 36 h. Then the slides were briefly rinsed with 1X SSC 0.1% SDS and washed once the same solution for 5 min. Three additional washes (one with 0.2XSSC 0.1% SDS and two with 0.2X SSC, for 5 min each) at room temperature were performed before drying the glass slides with a brief centrifugation. The probe design and these hybridisation/washing conditions allow the detection of specific genes even within gene families.
Scan protocol
The microarray was scanned with a two channel confocal microarray scanner (ScanArray® Lite, Perkin Elmer, USA) using its dedicated software (ScanArray Express 3.0.0., Perkin Elmer). The laser power and the photomultiplier tube (PMT) were set between 75 and 85 % of maximum. The excitation/emission settings were 543/570 nm for Cy3 and 633/670 nm for Cy5. After laser focusing and balancing of the two channels, scans were conducted at a resolution of 5 μm. For any scan, two separate 16-bit TIFF images were produced.
Description
Analysis used harvest fruit+1 day at room temperature RNA as control samples for comparison to the experimental samples taken after 6weeks at O°C + 1 day at room temperature.
Data processing
Software from the TM4 (www.tm4.org) package developed at TIGR (www.tigr.org, Saeed et al., 2003) was used to analyze microarray data. Images were processed using the Spotfinder 2.2.3. software by means of the Otsu algorithm. Spots were also visually examined to delete the non-uniform ones. The expression data extracted by Spotfinder were normalized by MIDAS 2.18 using the LOWESS (Locally Weighted Regression Scatter Plot Smoothing, Cleveland, 1979) algorithm with the “block mode”. Normalized split data were loaded in MeV 4.3 and subjected to SAM (Significance Analysis of Microarrays, Tusher et al., 2001) analyses. Since each comparison was repeated at least twice, and a “percentage cutoff filter”, set to 75%, was used to eliminate those gene with less than 3 data points, there were at least 3 values for each gene to be used in the SAM analysis. Lists of clones with significant changes in expression were identified at delta values that gave a false discovery rate (FDR) of about 5%.
