|
| Status |
Public on May 01, 2010 |
| Title |
Diploid vs tetraploid Ler-0 (line 1026-41) seedlings Replicate 5 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Total RNA from diploid Ler-0
|
| Organism |
Arabidopsis thaliana |
| Characteristics |
generation: established line
cell ploidy: diploid
cell line: Ler-0
tissue: seedlings
|
| Treatment protocol |
Seeds without recognizable emerging roots or which appeared to have germinated prematurely were discarded (less than 1%). The 6-8th leaves were harvested when the bud of the eleventh leaf was recognizable.
|
| Growth protocol |
Seeds were stratified for two days at 4ºC (on 0.8% agar) and transferred to a test chamber (HAEREUS) with constant light (~75 µmol photons/m2 sec) and 22ºC. About 100 seedlings per microarray analysis were harvested on the fifth day when root tips had penetrated the seed coat. Plants were grown on quartz sand and peaty mould (1:2 mixture) under constant light (~70 µmol photons/m2 sec), 40% relative humidity and 18º C in a Heareus (HEMZ 20/240/S) walk-in growth chamber equipped with OSRAM L58W-lamps (865-“LUMILUX Cool Daylight”, 530-“Warm White”, 77-“FLUORA”).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated using SVtotalRNA-System (Promega) as recommended by the supplier.
|
| Label |
Cy3
|
| Label protocol |
RNA (500ng) without detectable degradation and impurities was mixed with a spike-in mix, T7-promoter primer and denatured for 10 min followed by 2 hrs at 40ºC after addition of the cDNA master mix. The reaction was stopped at 65ºC for 15 min. The resulting cDNA was mixed with transcription master mix (with Cy3- or Cy5-label respectively) and incubated at 40ºC for additional 2 hrs. cRNA quantity and Cy3/Cy5-labelling efficiency was determined using a ND-1000 NanoDrop® spectrophotometer.
|
| |
|
| Channel 2 |
| Source name |
Total RNA from line 1026-41
|
| Organism |
Arabidopsis thaliana |
| Characteristics |
generation: F3
cell ploidy: tetraploid
cell line: 1026-41
tissue: seedlings
|
| Treatment protocol |
Seeds without recognizable emerging roots or which appeared to have germinated prematurely were discarded (less than 1%). The 6-8th leaves were harvested when the bud of the eleventh leaf was recognizable.
|
| Growth protocol |
Seeds were stratified for two days at 4ºC (on 0.8% agar) and transferred to a test chamber (HAEREUS) with constant light (~75 µmol photons/m2 sec) and 22ºC. About 100 seedlings per microarray analysis were harvested on the fifth day when root tips had penetrated the seed coat. Plants were grown on quartz sand and peaty mould (1:2 mixture) under constant light (~70 µmol photons/m2 sec), 40% relative humidity and 18º C in a Heareus (HEMZ 20/240/S) walk-in growth chamber equipped with OSRAM L58W-lamps (865-“LUMILUX Cool Daylight”, 530-“Warm White”, 77-“FLUORA”).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated using SVtotalRNA-System (Promega) as recommended by the supplier.
|
| Label |
Cy5
|
| Label protocol |
RNA (500ng) without detectable degradation and impurities was mixed with a spike-in mix, T7-promoter primer and denatured for 10 min followed by 2 hrs at 40ºC after addition of the cDNA master mix. The reaction was stopped at 65ºC for 15 min. The resulting cDNA was mixed with transcription master mix (with Cy3- or Cy5-label respectively) and incubated at 40ºC for additional 2 hrs. cRNA quantity and Cy3/Cy5-labelling efficiency was determined using a ND-1000 NanoDrop® spectrophotometer.
|
| |
|
| |
| Hybridization protocol |
cRNA (835ng) mixed with blocking agent and fragmentation buffer was incubated at RT (30 min) and fragmentation stopped with 2X hybridisation buffer. The mix was immediately loaded onto the microarray. Hybridization was performed at 65ºC for 17 hrs. The slides were subjected to three washing steps.
|
| Scan protocol |
Arrays were scanned in an Agilent Microarray Scanner G2565 (resolution: 5µm; channels: red & green; XDR: yes).
|
| Description |
Biological replicate 5
|
| Data processing |
Feature extraction and further processing was performed with the supplied Agilent feature extraction software (v.9.5.). Probe sequences of the Agilent Chip were realigned to cDNA sequences of the TAIR7 gene annotation requiring a complete match (100% identity) of the probe sequences to the respective transcript sequence. Normalization and analysis of differential gene expression was restricted to probes uniquely matching one particular TAIR7 gene locus. These probes are indicated by flag=1 in the sheet of the processed expression values of the matrix sheet. TAIR7 transcript names for each probe are shown in the 'name' column of the matrix sheet if one probe mapped uniquely and specifically one alternative splice variant of the respective locus, otherwise the TAIR7 locus identifier is reported for probes completely matching at least two alternative splice variants of the respective gene locus. Using the R/LIMMA package mean background intensities were subtracted from median red and green signals. M-values were normalized by the loess method for each array separately (“within array normalization”) and are shown as log2 Cy5/Cy3 ratios in the sample data tables.
|
| |
|
| Submission date |
Oct 08, 2009 |
| Last update date |
Oct 08, 2009 |
| Contact name |
Ramon Angel Torres-Ruiz |
| E-mail(s) |
[email protected]
|
| Phone |
+49-(0)8161-715643
|
| Fax |
+49-(0)8161-715636
|
| Organization name |
Technische Universität München, WZW
|
| Department |
Genetik
|
| Lab |
Torres-Ruiz
|
| Street address |
Emil-Ramann-Str. 8
|
| City |
Freising |
| ZIP/Postal code |
D-85354 |
| Country |
Germany |
| |
|
| Platform ID |
GPL6177 |
| Series (1) |
| GSE18482 |
Arabidopsis thaliana tetraploid transcriptome |
|
