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Adult mucous neck cells from corpus gastric epithelium

ABSTRACT: In this experiment, mucous neck cells from the gastric epithelium of normal, adult C57/B6 mice were laser-capture microdissected to determine gene expression in neck cells relative to pit cells, parietal cells, and zymogenic cells, whose expression profiles were previously deposited in GEO. In this screening experiment, only a single replicate Mouse Genome 430 2.0 GeneChip was generated from laser-capture microdissected cells in the neck region of the gastric unit (identified by positive staining with AlexaFluor-488-GS-II lectin positivity and/or position in the middle of the gastric unit with low labeling with E-cadherin and seconndary Alexafluor-488). RNA was purified by PicoPure kit, RNA integrity verified on Agilent Bioanalyzer, and total RNA pooled from multiple dissections and multiple mice to make ~100 ng RNA, which was then amplirifed, labeled, and fragmented (by Arctureus RiboAmp HS kit followed by the RNA Amplification and Labeling Kit from Enzo Life Sciences). Biotinylated cRNA probes were hybridized to the GeneChips. Given the inevitable contamination of the RNA with RNA from parietal cells, this GeneChip is used only in comparisons with laser-captured parietal cells described in GSE5018 as a baseline reference.

ORGANISM(S): Mus musculus

SUBMITTER: Jason Mills

PROVIDER: E-GEOD-42946 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Adult mucous neck cells from corpus gastric epithelium

Project description:In this experiment, mucous neck cells from the gastric epithelium of normal, adult C57/B6 mice were laser-capture microdissected to determine gene expression in neck cells relative to pit cells, parietal cells, and zymogenic cells, whose expression profiles were previously deposited in GEO.

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Project description:MIST1 is a bHLH transcription factor that is necessary for the maturation of gastric zymogenic cells as they differentiate from their precursor mucous neck cells. In this experiment, mucous neck cells and zymogenic cells of normal, adult C57BL/6 and MIST1 knockout mice were laser-capture microdissected in order to determine MIST1-dependent, zymogenic cell specific gene expression. Stomachs were excised immediately following sacrifice, quickly flushed with room-temperature PBS, inflated by duodenal injection of OCT compound, frozen in Cytocool II, and cut into serial 7-M-NM-<m-thick cryosections, which were mounted on Superfrost slides, fixed in 70% EtOH, rehydrated in nuclease-free water, and then incubated in Alexa Fluor 488-conjugated Griffonia simplicifolia GS-II (diluted 1:500 in nuclease-free water) for 15 min. Sections were washed in nuclease-free water and dehydrated in graded ethanol followed by xylene. ZCs were identified as corpus cells that were basal to GS-II labeling and which did not show the dark silhouettes and characteristic shape of parietal cells following xylene dehydration. Four wild-type mice and 5 Mist1M-bM-^HM-^R/M-bM-^HM-^R mice were used for dissection (PixCell II LCM apparatus [7.5-M-NM-<m spot diameter] and CapSure HS LCM caps) to generate two caps per mouse. RNA was purified by PicoPure kit, and RNA integrity was confirmed by an Agilent 2100 Bioanalyzer. All RNA from each cap was treated with DNase I and then reverse transcribed using the SuperScript III (Invitrogen) standard protocol (most cDNA syntheses started with 10 ng of total RNA). Biotinylated cRNA probes were hybridized to the GeneChips. Gene chip arrays used in these experiments were Affymetrix Mouse Gene 1.0ST arrays. Chip quality control and GeneChip to GeneChip comparisons to generate expression profiles were performed using dChip.

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