|
| Status |
Public on Apr 03, 2008 |
| Title |
Leaf_LCO treated_Rep 1_48 h After Treatment |
| Sample type |
RNA |
| |
|
| Source name |
First trifoliolate leaf, 48h after sprayed with lipo-chitooligosaccharide (NOD factor)
|
| Organism |
Glycine max |
| Characteristics |
Soybean cv. OAC Bayfield, V1 stage of development, first trifoliolate leaf.
|
| Treatment protocol |
Once the soybean plants had reached the V1 stage of development, the first trifoliolate leaf of each plant was sprayed with 2 mL of sterilized dH2O, containing 10-7 M LCO (lipo-chitooligosaccharide; NodBj-V (C18:1, MeFuc) from strain Bradyrhizobium japonicum strain 532C) and 0.02% Tween 20. 48 h later, the first trifoliolate leaf was harvested, immediately submerged in liquid N2, and stored at -80 ºC until extraction. LCO, 1 of 3 replicates.
|
| Growth protocol |
Soybean cultivar (cv.) Ontario Agricultural College (OAC) Bayfield seeds were surface-sterilized in 25% v/v sodium hypochlorite (Javex) for 2 min and then washed five times with distilled water (dH2O). The seeds were then spread evenly on a 5 cm thick bed of autoclaved vermiculite in a large plastic tray (54 x 28 x 6.5 cm) with holes at the bottom, to allow drainage of excess water, and then covered with 1 cm of vermiculite. The tray, with seeds, was placed in a growth chamber with a 16 h photoperiod (25 ºC) and an 8 h period of darkness (20 ºC). The germination/rooting medium was kept moist through regular additions of dH2O. When the cotyledons had emerged and separated (V1 growth stage) the seedlings were transferred to 5 cm pots containing a mixture of Pro-Mix (sphagnum peat moss) and perlite, as growth media. Each seedling was transferred to an individual pot and returned to the growth chamber under the conditions described above. The seedlings were watered with ½ strength Hoagland’s solution every day.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Frozen leaf tissue was rapidly ground with a mortar and pestle chilled with liquid N2 that had been placed in a -20 ºC freezer for 4 h prior to RNA extraction. Total RNA was extracted from ~100 mg of finely-ground, frozen tissue using the Qiagen RNeasy® Plant Mini Kit for total RNA isolation, with an on-column DNase digestion via the RNase-free DNase Set, according to the manufacturer’s instructions. Samples were stored at -80 oC until use.
|
| Label |
biotin
|
| Label protocol |
3.5 µg was used to synthesize labeled target cRNA using the GeneChip HT One-Cycle Target Labeling and Controls kit (Affymetrix). cRNA were labelled with biotin, stained with Streptavidin-phycoerythrin (SAPE) and detected with Biotinylated anti-streptavidin antibody.
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| |
|
| Hybridization protocol |
The cRNA quality was determined using a 2100 BioAnalyzer equipped with an RNA Nano LabChip, overnight hybridization of the chip utilized 15 µg of fragmented cRNA and the staining and washes were performed using an Affymetrix GeneChip Fluidics Station 450 robot, following the manufacturer's protocols.
|
| Scan protocol |
The microarray chips were scanned with an Affymetrix GeneChip Scanner 3000 7G, following the manufacturer's protocols.
|
| Description |
All steps followed Affymetrix guidelines and protocols.
|
| Data processing |
Robust multi-array average (RMA) analysis was performed on the .CEL files (with normalization and suppression of background noise). The base 2 logarithm of RMA signals were median-centered so that the median log-scale expression measure for each GeneChip was zero.
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| |
|
| Submission date |
Nov 29, 2007 |
| Last update date |
Aug 14, 2011 |
| Contact name |
John K Lindsay |
| E-mail(s) |
[email protected]
|
| Phone |
5143987851
|
| Organization name |
McGill University
|
| Department |
Plant Science
|
| Street address |
21111 Lakeshore Rd
|
| City |
Ste.Anne-de-Bellevue |
| State/province |
Quebec |
| ZIP/Postal code |
H9X 3V9 |
| Country |
Canada |
| |
|
| Platform ID |
GPL4592 |
| Series (1) |
| GSE9730 |
Effect of foliar spray of lipo-chitooligosaccharide (Nod factor) on soybean leaf gene expression. |
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