University of Illinois at Urbana-Champaign, Urbana IL
Manufacture protocol
Eight cesarean-derived piglets from two litters were randomly assigned to two GF isolators. Four piglets from one isolator were conventionalized with fecal slurry. On d 14, two cm long segments starting at the 85% of SI length measured distally of the pyloric sphincter were identified and embedded in Shandon Cryomatrix™ (Thermo Electron Corporation, Pittsburgh, PA). Microdissection and capture of villus and crypt cell populations were performed immediately on a Pixcell II ® LCM System (Arcturus) according to the manufacturer instructions. Two hundred to five hundred cells were collected onto a CapSure H LCM Cap (Arcturus) using the following parameters: spot size, 7.5 μm; power, 50 mW; pulse duration 2.00 s. Total RNA was isolated from laser captured villus and crypt cells using the PicoPureTM RNA isolation Kit (Arcturus) according to manufacturer’s recommendations. RNA from each sample was subjected to 2 rounds of amplification using the RiboAmpTM RNA linear amplification kit (Arcturus). Subsequently, RNA was reverse-transcribed into cDNA incorporating a T7 promoter. The cDNA was eluted with 16 μl of elution buffer and in vitro transcribed into amplified anti-sense RNA with a T7 RNA polymerase incorporating amino-allyl-UTP (Ambion, Austin, TX). The amplified anti-sense (aRNA) was labeled with Cy3 or Cy5 dyes. Each of the 16 cell populations was reversed labeled with Cy3 and Cy5 to account for dye labeling bias, resulting in 32 target samples for microarray hybridization. Porcine oligonucleotide set represents 13,297 porcine cDNAs and ESTs. A reference design was used in which each of the experimental samples was cohybridized with the reference sample that contained equal amounts of all the RNA samples used in the experiment. Single-spotted oligos were printed on GAPS II slides (Corning, Corning, NY) at the W. M. Keck Center for Comparative and Functional Genomics, University of Illinois. Following hybridization and washing, slides were scanned immediately for both dye channels with an Axon 4000B (Molecular Devices, Union City, CA) dual-laser confocal scanner and images were processed using GenePix v6.0 software (Molecular Devices).
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