The four RNA population from seedling roots, seedling shoots, panicles, and suspension-cultured cells were pooled in equal amount and cDNAs were derived from the RNA mixture. Total RNA and mRNA were isolated using the RNeasy Plant Mini kit (Qiagen, Valencia, CA) and the Oligotex mRNA kit (Qiagen), respectively. First-strand cDNA was generated from 4 µg poly(A)+ RNA with SuperScript II reverse transcriptase (Invitrogen, Carlsbad, CA). The cDNAs were precipitated in ethanol:isopropanol (1:1 v/v) and resuspended in 0.1 M NaHCO3 to facilitate coupling of Alexa Fluor 555 NHS esters (Molecular Probes, Eugene, OR) to the reactive groups of the amino-allyl dUTPs and were purified with CyScribe GFX glass fiber spin columns (Amersham Bioscience, Piscataway, NJ). Microarrays were hybridized with 4 µg labeled cDNA in 300 µL hybridization buffer (50 mM MES, 0.5 M NaCl, 10 mM EDTA, and 0.005% (v/v) Tween-20) for 16 hours at 50°C in disposable adhesive chambers (Grace BioLabs, Bend, OR) in a hybridization oven with constant agitation. Hybridized arrays were washed on an orbital platform in non-stringent buffer (6× SSPE, 0.01% [v/v] Tween-20) for 10 minutes at room temperature, then in stringent buffer (100 mM MES, 0.1 M NaCl, 0.01% Tween-20) for 30 minutes at 45°C. This was followed by a 5-minute wash in non-stringent buffer and a 2-minute wash in 0.1X SSC. The arrays were dried with compressed nitrogen and scanned with an Axon 4000B laser scanner at 5 µm resolution. The fluorescence intensity data were extracted with the NimbleScan software (NimbleGen Systems, Madison, WI). Keywords = Rice Keywords = poly(A)+ RNA
