|
| Status |
Public on Jun 27, 2011 |
| Title |
Seed (Se) vs Mesocarp (Me) at early development phase |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
seed early development (Se)
|
| Organism |
Prunus persica |
| Characteristics |
cultivar: Fantasia
tissue: seed
developmental stage: early
|
| Treatment protocol |
No treatment was performed.
|
| Growth protocol |
Peach of cv 'Fantasia’ were collected at 42, 60, 106 and 123 days after full bloom (DAFB), corresponding to the first exponential growth phase (S1), the onset (S2I) of pit hardening, the second exponential growth phase (S3) and the ripening (S4), respectively. Mesocarp and seed were excised from fruit and immediately frozen in liquid nitrogen and stored at -80°C until use. To monitor seed development, seeds were excised from fruit starting from late S1 up to ripening at weekly intervals, dissected and viewed by stereomicroscopy.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Frozen mesocarp and seed were ground in liquid nitrogen to a fine powder and total RNA was extracted as described by Ruperti and co-workers (2001, Physiol Plant 2001, 111(3):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 |
Mesocarp early development (Me)
|
| Organism |
Prunus persica |
| Characteristics |
cultivar: Fantasia
tissue: mesocarp
developmental stage: early
|
| Treatment protocol |
No treatment was performed.
|
| Growth protocol |
Peach of cv 'Fantasia’ were collected at 42, 60, 106 and 123 days after full bloom (DAFB), corresponding to the first exponential growth phase (S1), the onset (S2I) of pit hardening, the second exponential growth phase (S3) and the ripening (S4), respectively. Mesocarp and seed were excised from fruit and immediately frozen in liquid nitrogen and stored at -80°C until use. To monitor seed development, seeds were excised from fruit starting from late S1 up to ripening at weekly intervals, dissected and viewed by stereomicroscopy.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Frozen mesocarp and seed were ground in liquid nitrogen to a fine powder and total RNA was extracted as described by Ruperti and co-workers (2001, Physiol Plant 2001, 111(3):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 |
Cy3,Cy5
|
| 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 100-150 pmol of Cy3- and Cy5-labelled target cDNAs. The glass slides were placed in a hybridisation chamber (Corning, USA) kept on a water bath at 48°C 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 to compare transcript levels in seed (Se) and mesocarp (Me) at early development.
|
| 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”, keeping as reference the Cy3 channel. The “IA” and “IB” values have been used to determine log2(IB/IA) reported in the “VALUE column of the data table. The data from the technical replicates have been averaged.
Normalized split data were loaded in MeV 4.3 and subjected to SAM (Significance Analysis of Microarrays, Tusher et al., 2001) analyses. Lists of clones with significant changes in expression were identified at delta values that gave a false discovery rate (FDR) of 0% (90th percentile).
The labeling scheme for each channel of the supplementary files is available in the 'GSE22582_readme.txt' file, which is linked to the Series GSE22582 record.
|
| |
|
| Submission date |
Jul 01, 2010 |
| Last update date |
Jun 27, 2011 |
| Contact name |
claudio Bonghi |
| E-mail(s) |
claudio.bonghi@unipd.it
|
| Phone |
+390498272844
|
| Fax |
+390498272850
|
| Organization name |
University of Padova
|
| Street address |
Viale dell'UNiversità, 16
|
| City |
Legnaro |
| State/province |
Padova |
| ZIP/Postal code |
IT-35020 |
| Country |
Italy |
| |
|
| Platform ID |
GPL8584 |
| Series (1) |
| GSE22582 |
A microarray approach to identify genes involved in seed and mesocarp development in peach |
|
| Supplementary file |
Size |
Download |
File type/resource |
| GSM562205_Se_vs_Me_peach_12as.mev.gz |
106.0 Kb |
(ftp)(http) |
MEV |
| GSM562205_Se_vs_Me_peach_12as_MDS.mev.gz |
102.5 Kb |
(ftp)(http) |
MEV |
| GSM562205_Se_vs_Me_peach_12bs.mev.gz |
106.4 Kb |
(ftp)(http) |
MEV |
| GSM562205_Se_vs_Me_peach_12bs_MDS.mev.gz |
102.8 Kb |
(ftp)(http) |
MEV |
| GSM562205_Se_vs_Me_peach_20as.mev.gz |
114.4 Kb |
(ftp)(http) |
MEV |
| GSM562205_Se_vs_Me_peach_20as_MDS.mev.gz |
108.7 Kb |
(ftp)(http) |
MEV |
| GSM562205_Se_vs_Me_peach_20bs.mev.gz |
115.4 Kb |
(ftp)(http) |
MEV |
| GSM562205_Se_vs_Me_peach_20bs_MDS.mev.gz |
109.3 Kb |
(ftp)(http) |
MEV |
| GSM562205_Se_vs_Me_peach_22a.mev.gz |
133.6 Kb |
(ftp)(http) |
MEV |
| GSM562205_Se_vs_Me_peach_22a_MDS.mev.gz |
122.2 Kb |
(ftp)(http) |
MEV |
| GSM562205_Se_vs_Me_peach_22b.mev.gz |
131.8 Kb |
(ftp)(http) |
MEV |
| GSM562205_Se_vs_Me_peach_22b_MDS.mev.gz |
120.8 Kb |
(ftp)(http) |
MEV |
| Processed data included within Sample table |
| Processed data provided as supplementary file |
