|
| Status |
Public on Sep 01, 2007 |
| Title |
wt_dark24hr_vs_wt_FRc24hr_2 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
LEM0115
|
| Organism |
Solanum lycopersicum |
| Characteristics |
Solanum lycopersicum, Whole seed, Development
|
| Treatment protocol |
24hr FRc
|
| Growth protocol |
Incubation in distilled water-saturated cotton wool in clear plastic boxes cover with black plastic sheets at 25ºC
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Phenol method with carbohydrate-enriched tissue such as tubers and trizol for other tissues such as leaves (SGED_SOP_3.1.1 + SGED_SOP_3.3.1)
|
| Label |
Cy3
|
| Label protocol |
Amino allyl labeling and direct labeling (SGED_SOP_5.1.1 + SGED_SOP_6.1.1)
|
| |
|
| Channel 2 |
| Source name |
LEM0112
|
| Organism |
Solanum lycopersicum |
| Characteristics |
Solanum lycopersicum, Whole seed, Development
|
| Treatment protocol |
Darkness
|
| Growth protocol |
Incubation in distilled water-saturated cotton wool in clear plastic boxes cover with black plastic sheets at 25ºC
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Phenol method with carbohydrate-enriched tissue such as tubers and trizol for other tissues such as leaves (SGED_SOP_3.1.1 + SGED_SOP_3.3.1)
|
| Label |
Cy5
|
| Label protocol |
Amino allyl labeling and direct labeling (SGED_SOP_5.1.1 + SGED_SOP_6.1.1)
|
| |
|
| |
| Hybridization protocol |
Hybridization with indirectly labeled mRNA (SGED_SOP_6.1.1, SGED_SOP_6.2.1-4)
|
| Scan protocol |
Axon 4000B scanner. Both the 635nm (red, Cy5) and 532nm (green, Cy3) channels are scanned simultaneously at 100% laser power, the PMT set between 600 and 950. Slides are scanned at a resolution of 10micron
|
| Description |
Treatment of the seeds. The Solanum lycopersicum var. Money Maker wild type (MM) and fri1-1 mutant seeds were incubated on distilled-water saturated cotton wool in clear plastic boxes wrapped in black plastic sheets in darkness during 3 hours at 25°C. After that, the seeds were treated as follows: a) 24 hr of continuous irradiation with far-red light, FRc; b) 24 hr of FRc follow by a 10 min red light pulse (Rp), FRc+Rp; and c) complete darkness. After the light treatments, the seeds were incubated in darkness (25°C) until sampling. Red light (35umol/m2sec) was provided by 40W fluorescent lamps (Phillips 40/15) in combination with a red translucid acrylic. FRc light (100 umol/m2sec) was provided by a set of 150W incandescent internal reflector lamps (Phillips) in combination with a red acetate filter, six 2mm thick blue acrylic filters Paolini 2031 and 10cm water filter. Total RNA isolation. The seeds were grinded in liquid nitrogen and extracted with two volumes of extraction buffer (1% SDS, 6% p-aminosalycilic acid, 1% NaCl, 6% water-saturated phenol and 2% 2-mercaptoethanol) and two volumes of acid phenol:chloroform solution. The aqueous phase was sequentially extracted with one volume of acid phenol:chloroform solution and one volume of chloroform. The nucleic acids in the aqueous phase were precipitated with 0.15 volumes of 3M NaCl and 2.5 volumes of 100% ethanol at –80°C for 20 minutes. After centrifugation, the pellet was washed with 70% ethanol, resuspended in RNase-free water and precipitated with one volume of 4M LiCl over night at 4°C. The LiCl was removed after centrifugation and the pellet was rinsed with 2M LiCl and resuspended in RNase-free water. The total RNA was treated with RNase-free DNase (Promega) and cleaned up with mini spin columns (RNeasy Plant Mini Kit, Qiagen). The mRNA was amplificated with Message Amp II aRNA Amplification Kit (Ambion) and concentrated by vacumm centrifugation.
|
| Data processing |
The TIFF images were quantified using Genepix 5.0. Local background was subtracted from the signal value and the data was normalized using the quantile method in the limma package of bioconductor.
|
| |
|
| Submission date |
Jun 22, 2007 |
| Last update date |
Aug 14, 2011 |
| Contact name |
Jia Liu |
| E-mail(s) |
[email protected]
|
| URL |
http://www.tigr.org/tdb/potato
|
| Organization name |
Plant Genomics
|
| Street address |
9712 Medical Center Drive
|
| City |
Rockville |
| State/province |
MD |
| ZIP/Postal code |
20850 |
| Country |
USA |
| |
|
| Platform ID |
GPL3838 |
| Series (1) |
| GSE8246 |
Global analysis of gene expression associated with germination in tomato seeds. |
|
| Data table header descriptions |
| ID_REF |
Spot identifier for each feature |
| VALUE |
Normalized log2 ratio of normalized intensities defined by CH2/CH1. This value is set to null if it is flagged with "M" or "X" |
| CH1_NORMALIZED |
Normalized background subtracted CH1 intensity (RED channel) |
| CH1_RAW |
Background (CH1_BACKGROUND) subtracted raw intensity (F635 Mean - B635 Media) |
| CH1_BACKGROUND |
CH1 background median intensity (B635 Media) |
| CH2_NORMALIZED |
Normalized background subtracted CH2 intensity (GREEN channel) |
| CH2_RAW |
Background (CH2_BACKGROUND) subtracted raw intensity (F532 Mean - B532 Media) |
| CH2_BACKGROUND |
CH2 background median intensity (B532 Media) |
| FLAG |
B: no flag, good spot; X: undetectable spot; M: flagged for diameter < 70 microns, the percentage of saturated pixels > 30% or not validated PCR product |
