|
| Status |
Public on Jan 01, 2014 |
| Title |
Ler-control_rep2 |
| Sample type |
RNA |
| |
|
| Source name |
Ler ecotype, control, replicate 2
|
| Organism |
Arabidopsis |
| Characteristics |
tissue: leaf
ecotype: Ler
|
| Treatment protocol |
Ten days after sowing, uniform seedlings of both ecotypes were carefully transferred to MS plates supplemented with 100 mM NaCl. Seedlings in MS plates without salt were used as controls. Plant materials were collected for RNA extraction at 4th days after transfer.
|
| Growth protocol |
Seeds of Sha and Ler ecotypes were sown in MS plates and grown at 22/18 °C in the growth chamber under a photoperiod of 12h light/12 h dark at 350 μmol m-2s-1 and 70% relative humidity.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted and purified from leaves of at least 30 seedlings per plate for each ecotype and salt combination using QIAGEN-RNeasy Mini Kit (Qiagen, Valencia, CA, USA) according to guidelines specified by the manufacturer. Two biological replicates were prepared for each combination. RNA was quantified using a Nanodrop-ND 8000 spectrophotometer (Thermo Fisher Scientific) and RNA quality was assessed by agarose gel analysis and using a 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) according to the manufacturer’s protocol. Samples with the RNA integrity number of 7.5 and above were chosen for further analysis.
|
| Label |
cy3
|
| Label protocol |
Microarray analysis was performed using Agilent-021169 Arabidopsis 4 Oligo Microarray (V4) (Probe Name version). In total 150ng of total RNA was used to prepare Cyanine-3 (Cy3) labeled probe with the help of low RNA input linear amplification/labeling kit (Agilent technologies) and 1.65µg of Labeled cRNA probes were fragmented using fragmentation buffer (Agilent Technologies) and hybridized to the Arabidopsis arrays according to manufacturer’s instructions.
|
| |
|
| Hybridization protocol |
Microarray analysis was performed using Agilent-021169 Arabidopsis 4 Oligo Microarray (V4) (Probe Name version). In total 150ng of total RNA was used to prepare Cyanine-3 (Cy3) labeled probe with the help of low RNA input linear amplification/labeling kit (Agilent technologies) and 1.65µg of Labeled cRNA probes were fragmented using fragmentation buffer (Agilent Technologies) and hybridized to the Arabidopsis arrays according to manufacturer’s instructions.
|
| Scan protocol |
The arrays were scanned using the high resolution array scanner (Agilent technologies) with the appropriate settings for the one color gene expression arrays after hybridization.
|
| Data processing |
Array images were acquired with Agilent's dual-laser microarray scanner and signal intensities were extracted from the scanned images with dedicated Agilent Feature Extraction software (Agilent technologies). The outliers and the abnormal features were flagged and the data was normalized using the intra-array percentile shift normalization (threshold of 75 and above) and median based inter-array normalization. The GeneSpring software (Agilent technologies) was used to calculate the intensity ratios and fold changes.
All the genes with a P value below 0.01 and a fold change above 2 were chosen for further analysis.
|
| |
|
| Submission date |
Sep 18, 2012 |
| Last update date |
Jan 01, 2014 |
| Contact name |
Zhulong Chan |
| E-mail(s) |
zlchan@mail.hzau.edu.cn
|
| Organization name |
Huazhong Agricultural University
|
| Department |
College of Horticulture and Forestry Sciences
|
| Street address |
No. 1, Shizishan St, Nanhu
|
| City |
Wuhan |
| State/province |
Hubei Province |
| ZIP/Postal code |
430070 |
| Country |
China |
| |
|
| Platform ID |
GPL9020 |
| Series (1) |
| GSE40940 |
Natural variation of transcriptional networks in Arabidopsis thaliana in response to salt stress |
|
