|
| Status |
Public on Oct 22, 2010 |
| Title |
0 vs. 15 days. Replicate 2 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Seeds of Pisum sativum L. cv Alaska Early (‘Abundant Life Seed Foundation’, Port Townsend, USA)
|
| Organism |
Pisum sativum |
| Characteristics |
cultivar: Alaska Early
tissue: Seeds
age: Non-aged control
|
| Treatment protocol |
Seeds of Pisum sativum L. cv Alaska Early (‘Abundant Life Seed Foundation’, Port Townsend, USA) were equilibrated at 20 ± 1 °C for ~5 weeks over non-saturated LiCl of 60% relative humidity (Hay et al., 2008) until their MC was stable at 12%. Relative humidity was recorded with a Rotronic AWVC-D10 Hygropalm and MC measured gravimetrically after drying for 17 h at 103 °C. These equilibrated seeds are referred to as ‘non-aged controls’. Seeds of 12 % MC were artificially aged in sealed bottles at 50 °C. Viability was lost after 55 d, during which time seed MC did not change significantly. At intervals, germination tests were performed on 1 % water agar at 25 °C at a day (15 umol m-2 s-1) / night cycle of 8 /16h. Germination was defined as radical emergence by >2 mm. For molecular analyses, 3-5 replicates of 20 pea seeds each were taken at each ageing interval, immediately frozen in liquid nitrogen, freeze dried and ground to a fine powder in hermetically closed, liquid nitrogen-cooled Teflon capsule using a laboratory mill (Braun Microdismembrator). The powder was stored at - 70 °C in humidity-proof vials until use in all assays
|
| Growth protocol |
Germination tests were performed on 1 % water agar at 25 °C under warm white fluorescent light (15 umol m-2 s-1) at a day / night cycle (8 /16h). Germination was defined as radical emergence by at least 2 mm.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated from 30-50 mg ground seed powder following protocols of Oñate-Sánchez and Vicente-Carbajosa (2008)
|
| Label |
Cy3
|
| Label protocol |
Total RNA (1 µg of each) was amplified and aminoallyl-labelled using MessageAmpTM II aRNA kit (Ambion) and 5-(3-aminoallyl)-2’deoxyuridine-5’-triphosphate (aa-dUTP, Ambion) following the manufacturer´s instructions. Approximately, 40-50 µg of aRNA was obtained. For each sample, 7.5 µg of aminoallyl-labelled aRNA was resuspended in 0.1 M Na2CO3 (pH 9.0) and labelled with either Cy3 or Cy5 Mono NHS Ester (CyTMDye Post-labelling Reactive Dye Pack, Amersham). The samples were purified following the manufacturer´s instructions for MegaclearTM (Ambion). Cy3 and Cy5 incorporation was measured using 1 µl of the probe in a Nanodrop spectrophotometer (Nanodrop Technologies Inc.). For each hybridization 200 pmol of Cy3 and Cy5 probes were mixed, dried in speed-vac, and resuspended in 9 µl of RNase-free water. Labelled aRNA was fragmented by adding 1 µl of 10X Fragmentation buffer (Ambion) and incubating at 70ºC for 15 minutes. The reaction was stopped with 1 µl of Stop solution (Ambion). Integrity and average size of Total RNA, aRNA and fragmented aRNA were evaluated using a Bioanalyzer 2100 (Agilent). Average size of aRNAs was about 1000 nucleotides and of fragmented aRNAs 100 nucleotides. The final volume of the probe was diluted to 100 µl in hybridization solution.
|
| |
|
| Channel 2 |
| Source name |
Seeds of Pisum sativum L. cv Alaska Early (‘Abundant Life Seed Foundation’, Port Townsend, USA)
|
| Organism |
Pisum sativum |
| Characteristics |
cultivar: Alaska Early
tissue: Seeds
age: Aged 15 days
|
| Treatment protocol |
Seeds of Pisum sativum L. cv Alaska Early (‘Abundant Life Seed Foundation’, Port Townsend, USA) were equilibrated at 20 ± 1 °C for ~5 weeks over non-saturated LiCl of 60% relative humidity (Hay et al., 2008) until their MC was stable at 12%. Relative humidity was recorded with a Rotronic AWVC-D10 Hygropalm and MC measured gravimetrically after drying for 17 h at 103 °C. These equilibrated seeds are referred to as ‘non-aged controls’. Seeds of 12 % MC were artificially aged in sealed bottles at 50 °C. Viability was lost after 55 d, during which time seed MC did not change significantly. At intervals, germination tests were performed on 1 % water agar at 25 °C at a day (15 umol m-2 s-1) / night cycle of 8 /16h. Germination was defined as radical emergence by >2 mm. For molecular analyses, 3-5 replicates of 20 pea seeds each were taken at each ageing interval, immediately frozen in liquid nitrogen, freeze dried and ground to a fine powder in hermetically closed, liquid nitrogen-cooled Teflon capsule using a laboratory mill (Braun Microdismembrator). The powder was stored at - 70 °C in humidity-proof vials until use in all assays
|
| Growth protocol |
Germination tests were performed on 1 % water agar at 25 °C under warm white fluorescent light (15 umol m-2 s-1) at a day / night cycle (8 /16h). Germination was defined as radical emergence by at least 2 mm.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated from 30-50 mg ground seed powder following protocols of Oñate-Sánchez and Vicente-Carbajosa (2008)
|
| Label |
Cy5
|
| Label protocol |
Total RNA (1 µg of each) was amplified and aminoallyl-labelled using MessageAmpTM II aRNA kit (Ambion) and 5-(3-aminoallyl)-2’deoxyuridine-5’-triphosphate (aa-dUTP, Ambion) following the manufacturer´s instructions. Approximately, 40-50 µg of aRNA was obtained. For each sample, 7.5 µg of aminoallyl-labelled aRNA was resuspended in 0.1 M Na2CO3 (pH 9.0) and labelled with either Cy3 or Cy5 Mono NHS Ester (CyTMDye Post-labelling Reactive Dye Pack, Amersham). The samples were purified following the manufacturer´s instructions for MegaclearTM (Ambion). Cy3 and Cy5 incorporation was measured using 1 µl of the probe in a Nanodrop spectrophotometer (Nanodrop Technologies Inc.). For each hybridization 200 pmol of Cy3 and Cy5 probes were mixed, dried in speed-vac, and resuspended in 9 µl of RNase-free water. Labelled aRNA was fragmented by adding 1 µl of 10X Fragmentation buffer (Ambion) and incubating at 70ºC for 15 minutes. The reaction was stopped with 1 µl of Stop solution (Ambion). Integrity and average size of Total RNA, aRNA and fragmented aRNA were evaluated using a Bioanalyzer 2100 (Agilent). Average size of aRNAs was about 1000 nucleotides and of fragmented aRNAs 100 nucleotides. The final volume of the probe was diluted to 100 µl in hybridization solution.
|
| |
|
| |
| Hybridization protocol |
Cy5 and Cy3 amplified RNA (aRNA) fragmented probes were mixed (200 pmol of each label) with 20 µg of PolyA (Sigma) and 20 µg of yeast tRNA (Sigma) in a final volume of 90 µl of hybridization buffer (50% formamide, 6X SSC, 0.5% SDS, 5X Denhardt’s). The probe was denatured at 95ºC for 5 minutes and applied to the slide using a LifterSlip (Erie Scientific). Slides were then incubated at 42ºC for 16h in hybridization chambers (Array-It). After incubation, slides were washed twice with 0.5X SSC, 0.1% SDS for 5 minutes each, twice with 0.5X SSC for 5 minutes and finally in 0.05X SSC for 5 minutes. Slides were dried by centrifugation at 563g for 1 minute.
|
| Scan protocol |
Images from Cy3 and Cy5 channels were equilibrated and captured with a GenePix 4000B (Axon) and spots quantified using GenPix software (Axon).
|
| Description |
Biological replicate 2 of 3. Control non-aged seeds vs. 15 days aged seeds.
|
| Data processing |
Background correction and normalization of expression data were performed using LIMMA (Smyth and Speed, 2003; Smyth, 2004). LIMMA is part of Bioconductor, an R language project (Ihaka and Gentleman, 1996). First, the data set was filtered based on the spot quality. A strategy of adaptive background correction was used that avoids exaggerated variability of log-ratios for low-intensity spots. For local background correction the normexp method in LIMMA to adjust the local median background was used. The resulting log-ratios were print-tip loess normalized for each array (Smyth and Speed, 2003). To have similar distribution across arrays and to achieve consistency among arrays, log-ratio values were scaled using as scale estimator the median-absolute-value (Smyth and Speed, 2003). Linear model methods were used for determining differentially expressed genes. Each probe was tested for changes in expression over replicates by using an empirical Bayes moderated t-statistic (Smyth, 2004). To control the false discovery rate p-values were corrected by using the method of Benjamani and Hochberg (1995). The expected false discovery rate was controlled to be less than 5% or 10% where specified. Only reproducible expression values (present in at least two gene replicates and FDR<0.05) were considered and normalized log2 ratios were averaged.
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| |
|
| Submission date |
Oct 21, 2010 |
| Last update date |
Oct 21, 2010 |
| Contact name |
Daniel Osuna |
| E-mail(s) |
daniel.osuna@usal.es
|
| Phone |
34-923-294500
|
| Fax |
34-923-294682
|
| Organization name |
Centro Hispano-Luso de Investigaciones Agrarias.
|
| Department |
Fisiología Vegetal, Universidad de Salamanca.
|
| Lab |
7
|
| Street address |
Río Duero, 12
|
| City |
Villamayor |
| State/province |
Salamanca |
| ZIP/Postal code |
37185 |
| Country |
Spain |
| |
|
| Platform ID |
GPL8288 |
| Series (1) |
| GSE24864 |
Seeds of Pisum sativum L. cv Alaska Early: non-aged controls vs. Aged |
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