The tissue sampled was the 1.5 cm tip of all crown roots that had developed at the time of harvest, representing the most metabolically active region based on whole-tissue visualization of triphenyl tetrazolium chloride staining.
Growth protocol
Plants were germinated and grown in Turface MVP contained in DeepotTM plastic conical pot and tray systems (Stuewe and Sons, Tangent, OR (D25L [pots], D50T [trays])). Seeds were planted approximately 2cm below the top of the tube/Turface MVP and germinated for one week in diH2O submerged so that the seeds were just above the water line. After germination, the water was drained and a media pumping system was initiated. A humidity cover was in place from the initial germination until plants were approximately 6 inches tall. Modified Hoagland’s Solution (1mM KH2PO4, 2.5mM KNO3, 1.25mM Ca(NO3)2, 1mM MgSO4, 0.75mM CaCl2, SPRINT330 such that 0.006mM with respect to Iron, 0.03mM FeSO4, 1μM H3Bo3, 1μM MnCl2, 1μM ZnSo4, 1μM CuSO4, 1μM NaMoO4) was pumped into the tanks until the tubes were submerged but the vegetative growth remained dry. The nutrient solution remained in the tank for approximately 3 minutes before draining. The submersion/drainage system repeated every 3 hours for the 3 weeks following germination. Growth chamber conditions were maintained at 50% humidity with a 16h day (26 C) and an 8h night (22 C). Light levels ranged from approximately 350 μM/m2/s at the base of the plant to -500 μM/m2/s at the top of the leaves at the time of harvest.
Extracted molecule
total RNA
Extraction protocol
Total RNA was isolated from frozen ground tissue (SQ Tissue Kit, Omega Biotek) and treated with DNase-I followed by polyA RNA isolation (Illustra mRNA Purification Kit, GE Biosciences) for all samples. The total RNA and polyA RNA samples were visualized and quantified on Agilent’s Bioanalyzer to check for degradation and to determine the concentration.
Label
Cy5
Label protocol
Each mRNA sample was made into double stranded DNA, amplified by an in-vitro transcription reaction and labeled with Cy3 or Cy5 fluorescent dyes using Agilent’s Low RNA Linear Amp Kit.
Hybridization protocol
Standard Agilent Hybridization protocol
Scan protocol
After hybridization, the microarray slides were washed and scanned immediately with Agilent’s G2505B DNA Microarray Scanner at two laser power settings (100% and 10%). The images were inspected visually for image artifacts, and feature intensities were extracted, filtered, and normalized with Agilent’s Feature Extraction Software (v 9.5.1).
Description
A0210265CR015 raw data filename: US23502373_251553810325_S01_GE2-v5_95_Feb07_1_1_cy5.txt Gene Expression at Tip (~2 cm) of crown roots from 4-week-old SX19 Syn10 DH, B73 and Mo17 seedlings grown in turface (Hoagland with 5 mM nitrate) will be harvested, frozen in liquid nitrogen and stored at -80C. The tissues will be grinded before sending to Mary/Brian. Agilent array: two dyes, no dye swap. Tip (~2 cm) of crown roots from 4-week-old SX19 Syn10 DH, B73 and Mo17 seedlings grown in turface (Hoagland with 5 mM nitrate) will be harvested, frozen in liquid nitrogen and stored at -80C. The tissues will be grinded before sending to Mary/Brian. Agilent array: two dyes, no dye swap.
Data processing
A multidimensional, weighted least-squares method was used to obtain normalization parameters for the data based on affine transformations. Two-channel hybridizations were considered as single channel. Therefore, the same raw data file is linked to two Samples appended as _Cy3 and _Cy5. Data Normalized as described via multidimensional, weighted least-squares method
