PolyA RNA was isolated from 400 ug of total leaf RNA using the Poly(A)Purist MAG kit from Ambion (Austin, TX). Two ug of polyA was transcribed into cDNA and fluorescently labeled with Cy3 and Cy5 dyes using the SuperScript Indirect cDNA Labeling System (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. Labeled cDNA for experimental (Cy5) and reference (Cy3) samples were combined, ethanol precipitated, and resuspended in 10 ul of water. The cDNA was heated at 100°C for 3 min before the addition of 70 ul of SlideHyb Glass Array Hybridization Buffer #1 (Ambion, Austin, TX). The hybridization solution was pipetted onto slides covered with a gapped coverslip (LifterSlips, Erie Scientific Company, Portsmouth, NH). Slides were incubated overnight at 42°C in Corning hybridization chambers, then washed in 1x SSC, 0.1% (w/v) SDS for 5 min at 42°C followed by 5 min washes in 0.1x SSC, 0.1% SDS (w/v); and 0.05x SSC at room temperature. Slides were scanned using the ScanArray 4000 Microarray Analysis System. Based on an initial low resolution (50 um) scan, laser power settings were manually adjusted to balance the intensities between the two channels and slides were rescanned at 10 um resolution. Typical laser settings were: Cy3 laser power=100, PMT=80; Cy5: laser power=85, PMT=75. GPR files were generated using GenePix Pro 4.1 software and the mean pixel intensities for each spot were used in subsequent data analysis. Data files were imported into GeneTraffic Duo Version 2.6 and data for each hybridization were normalized using the Lowess Sub-Grid method. Spots flagged by the program under the default settings (spots with a raw pixel intensity less than 100 or lower than the average background and spots with an intensity/background intensity ratio <1) were removed from further analysis. Keywords = Medicago Keywords = alfalfa Keywords = isoflavone synthase Lot batch = Batch 1_9
