|
| Status |
Public on May 31, 2014 |
| Title |
Chip-32, RB_Day16_BlueRep3 & RB_Day14_RedRep3 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
16-day dehydration-stressed root buds
|
| Organism |
Euphorbia esula |
| Characteristics |
biotype: 1984-ND001
tissue: root bud
treatment: dehydration stress
treatment time: 16 days
|
| Treatment protocol |
A population of leafy spurge plants were propagated through random cuttings from the genetically uniform biotype and maintained in a greenhouse. For dehydration studies, water was withheld from paradormant plants for 0, 3, 7, 14, and 21 days, and each treatment included three replicates for crown buds, and two/three replicates for root buds consisting of 40 plants per replicate. Relative water contents of aerial tissue and soil moisture levels were determined as previously described by Doğramacı et al. (2011). At the end of each dehydration treatment crown and root buds were collected from 34 plants, and samples were stored at -80OC. Additionally, at the end of each dehydration treatment,the aerial portions of remining six plants from each replicate were decapitated at the soil surface and vegetative growth from crown buds was monitored under growth-conducive conditions by re-hydrating the root system.
|
| Growth protocol |
A population of leafy spurge plants were propagated through random cuttings from the genetically uniform biotype 1984-ND001 and maintained in a greenhouse as described by Anderson and Davis (2004). Shoot cuttings were grown in No. 1 Ray Leach Cone-tainers (Stuewe and Sons Inc. Corvallis, OR, USA) in the greenhouse for three months prior to application of experimental treatments. The greenhouse was set at 25°C with a 16 h:8 h day:night photoperiod. Average daily light fluencies were approximately 350 μmol m-2 s-1 and 400 W high-pressure sodium lamps were used as to extend the day-length.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
At the end of each treatment, crown and root bud samples were collected, flash frozen in liquid N2, and stored at -80°C until RNA extraction. Crown and root bud samples were ground to a fine powder in liquid N2, and total RNA was extracted from approximately one gram of tissue according to the pine tree RNA extraction protocol (Chang et al. 1993). RNA quality and quantity was confirmed by spectrophotometry and agarose gel electrophoresis.
|
| Label |
Cy5
|
| Label protocol |
For microarray hybridizations, labeled cDNAs were prepared from 30 µg of total RNA using the Alexa Fluor cDNA labeling kit (Invitrogen, Carlsbad, CA, USA) according to manufacturer's protocols.
|
| |
|
| Channel 2 |
| Source name |
14-day dehydration-stressed root buds
|
| Organism |
Euphorbia esula |
| Characteristics |
biotype: 1984-ND001
tissue: root bud
treatment: dehydration stress
treatment time: 14 days
|
| Treatment protocol |
A population of leafy spurge plants were propagated through random cuttings from the genetically uniform biotype and maintained in a greenhouse. For dehydration studies, water was withheld from paradormant plants for 0, 3, 7, 14, and 21 days, and each treatment included three replicates for crown buds, and two/three replicates for root buds consisting of 40 plants per replicate. Relative water contents of aerial tissue and soil moisture levels were determined as previously described by Doğramacı et al. (2011). At the end of each dehydration treatment crown and root buds were collected from 34 plants, and samples were stored at -80OC. Additionally, at the end of each dehydration treatment,the aerial portions of remining six plants from each replicate were decapitated at the soil surface and vegetative growth from crown buds was monitored under growth-conducive conditions by re-hydrating the root system.
|
| Growth protocol |
A population of leafy spurge plants were propagated through random cuttings from the genetically uniform biotype 1984-ND001 and maintained in a greenhouse as described by Anderson and Davis (2004). Shoot cuttings were grown in No. 1 Ray Leach Cone-tainers (Stuewe and Sons Inc. Corvallis, OR, USA) in the greenhouse for three months prior to application of experimental treatments. The greenhouse was set at 25°C with a 16 h:8 h day:night photoperiod. Average daily light fluencies were approximately 350 μmol m-2 s-1 and 400 W high-pressure sodium lamps were used as to extend the day-length.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
At the end of each treatment, crown and root bud samples were collected, flash frozen in liquid N2, and stored at -80°C until RNA extraction. Crown and root bud samples were ground to a fine powder in liquid N2, and total RNA was extracted from approximately one gram of tissue according to the pine tree RNA extraction protocol (Chang et al. 1993). RNA quality and quantity was confirmed by spectrophotometry and agarose gel electrophoresis.
|
| Label |
Cy3
|
| Label protocol |
For microarray hybridizations, labeled cDNAs were prepared from 30 µg of total RNA using the Alexa Fluor cDNA labeling kit (Invitrogen, Carlsbad, CA, USA) according to manufacturer's protocols.
|
| |
|
| |
| Hybridization protocol |
Labeled cDNAs were hybridized to a custom-made 23K element microarray that contained 19,808 UniGenes from a leafy spurge EST database (Anderson et al. 2007) and an additional 4,129 UniGenes from a cassava EST database (Lokko et al. 2007). Comparison of gene expression between samples was accomplished using a rolling circle dye-swap hybridization scheme (Churchill 2002) to provide every biological replicate with technical replicates. In this experiment, each of the biological replicates included two technical replicates.
|
| Scan protocol |
Microarray hybridization was visualized using a GenePix 4000B scanner and probe intensities and background were quantified using GenePix 6.0 software (Molecular Devices, Sunnyvale, California, USA).
|
| Description |
See Dogramaci et al. Plant Molecular Biology (2014) for more details.
|
| Data processing |
A quality control value of “1” was assigned to all probes that had intensity values greater than 2 times the standard deviation over average of the negative control and empty probe intensities (after deleting 1% of the most intense negative/empty probe values). Intensity values of control spots (a total of 1455 spots including 3X SSC, blanks, and mouse) from MA chips were removed before bioinformatics. In this submission, the original “GPR” files include information for the control spots, while the Sample data tables include information only for the spots that were used for data analyses (a total of: 23937). Raw signal intensities of all DNA-containing spots were up-loaded into the GeneMaths XT 5.1 microarray data analysis program (Applied Maths Inc., Austin, TX, USA) for normalization following the Layer/Normalization/Arrays path with default settings (Offset: Average; Scaling: Standard deviation), and for statistical analysis and clustering of the dataset. Hybridization intensities were log2 transformed, and arrays were centered and normalized against each other; technical replicates within each biological replicate were averaged.
|
| |
|
| Submission date |
Feb 18, 2014 |
| Last update date |
May 31, 2014 |
| Contact name |
Munevver Dogramaci |
| E-mail(s) |
Munevver.Dogramaci@ars.usda.gov
|
| Phone |
701-2391292
|
| Organization name |
USDA-ARS
|
| Street address |
1605 Albrecht Blvd N
|
| City |
Fargo |
| State/province |
ND |
| ZIP/Postal code |
58102 |
| Country |
USA |
| |
|
| Platform ID |
GPL4655 |
| Series (1) |
| GSE55133 |
Dehydration-induced endodormancy in crown buds of leafy spurge highlights roles for an RVE1-like homolog and hormone signaling cross-talk |
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