Mouse liver mRNA was isolated from adult Swiss Webster liver tissue obtained from Pel-Freez Biologicals (Rogers, AK). mRNA isolation was performed using oligo-dT hybrid capture on magnetic streptavidin beads using a commercially available kit (PolyATract System 1000, Promega, Madison, WI). poly A+ RNA was converted to double-stranded cDNA using GibCo (Rockville, MD) BRL’s SuperScript Choice System and an oligo dT primer containing the T7 RNA polymerase promoter (5’-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-T24-3’). in vitro transcription (IVT) was used to produce biotin-labeled cRNA from the cDNA using the Ambion (Austin, TX) MEGAscript T7 kit. cRNA was purified using Qiagen RNeasy RNA purification columns according to the manufacturer’s instructions. Before hybridization, cRNA was fragmented to an average size of 50-200 bp. Microarrays were hybridized with 12 ug cRNA in 300 ul, in the presence of 50 mM MES, 0.5 M NaCl, 10 mM EDTA, and 0.005% (v/v) Tween-20 for 16 hours at 45 degrees C. After hybridization, arrays were washed in non-stringent (NS) buffer (6X SSPE, 0.01% (v/v) Tween 20) for 5 min at RT, followed by washing in stringent buffer (100 mM MES, 0.1 M NaCl, 0.01% Tween 20) for 30 min at 45 degrees C. After washing, arrays were stained with streptavidin-Cy3 conjugate from Amersham Pharmacia for 25 min at RT, followed by a 5 min wash in NS buffer, a 30 sec rinse in 1X NimbleGen final rinse buffer (NimbleGen), and a blow-dry step using high-pressure grade-5 Argon (Badger Welding, Madison, WI). Arrays were scanned using an Axon 4000B scanner at 5 um resolution . Prior to data extraction, images were rotated and doubled in size (without interpolation) using ImageJ software (http://rsb.info.nih.gov/ij/). Features were extracted using GenePix 3.0 software (Axon Instruments, Inc.; Union City, CA), using a fixed feature size. The local background correction from the GenePix software was not applied to raw signal intensities. The values presented are average raw signal intensities.
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These data are being made available for research purposes only. NimbleGen Systems Inc. does not warrant or assume any legal liability or responsibility for the accuracy, completeness, or usefulness of this data for any purpose.
