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In situ-synthesized novel microarray optimized for mouse stem cell and early developmental expression profiling: Part 3

ABSTRACT: E12.5 mouse whole embryos and placentas were pooled within 3 litters and total RNA was extracted. Fluorescently-labeled, linearly-amplified cRNA targets were prepared from the RNA samples at various input levels. A "full-scale" reference was prepared using the standard input level of 6.0 ug total RNA, and one round of amplification was performed with 250 ng and 50 ng total RNA inputs. For 10 ng and 2 ng inputs, two rounds of amplification were used. Targets were compared on a microarray platform designed for this test using a "dye-swapped" experimental design, and sets of significantly differential genes were compared to the full scale reference's list to determine specificity and sensitivity in terms of detection of significant expression differences. Keywords: dye swap design,quality control testing design E12.5 mouse whole embryos and placentas were pooled within 3 litters and total RNA was extracted. Fluorescently-labeled, linearly-amplified cRNA targets were prepared from the RNA samples at various input levels. A "full-scale" reference was prepared using the standard input level of 6.0 ug total RNA, and one round of amplification was performed with 250 ng and 50 ng total RNA inputs. For 10 ng and 2 ng inputs, two rounds of amplification were used. Targets were compared on a microarray platform designed for this test using a "dye-swapped" experimental design, and sets of significantly differential genes were compared to the full scale reference's list to determine specificity and sensitivity in terms of detection of significant expression differences.

ORGANISM(S): Mus musculus

SUBMITTER: Kazuhiro Aiba

PROVIDER: E-GEOD-3507 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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In situ-synthesized novel microarray optimized for mouse stem cell and early developmental expression profiling.

Carter Mark G MG Hamatani Toshio T Sharov Alexei A AA Carmack Condie E CE Qian Yong Y Aiba Kazuhiro K Ko Naomi T NT Dudekula Dawood B DB Brzoska Pius M PM Hwang S Stuart SS Ko Minoru S H MS

Genome research 20030501 5

Applications of microarray technologies to mouse embryology/genetics have been limited, due to the nonavailability of microarrays containing large numbers of embryonic genes and the gap between microgram quantities of RNA required by typical microarray methods and the miniscule amounts of tissue available to researchers. To overcome these problems, we have developed a microarray platform containing in situ-synthesized 60-mer oligonucleotide probes representing approximately 22,000 unique mouse t ...[more]

PMID: 12727912

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Project description:BACKGROUND & AIMS: Gastric cancer results from a combination of Helicobacter pylori (H pylori) infection, exposure to dietary carcinogens, and predisposing genetic make-up. Because the role of these factors in gastric carcinogenesis cannot be determined readily in human beings, the present study examined the role of an oral carcinogen and H pylori infection in rhesus monkeys. METHODS: Gastroscopies were performed in 23 monkeys assigned to 4 groups: controls; nitrosating carcinogen ethyl-nitro-nitrosoguanidine administration alone; and inoculation of a virulent H pylori strain alone (H), or in combination with ethyl-nitro-nitrosoguanidine (EH). Follow-up gastroscopies and biopsies were performed at 3-month intervals for 5 years for pathologic and molecular studies. RESULTS: Postinoculation, H and EH groups showed persistent infection and antral gastritis. Starting at 2 and 5 years, respectively, gastric intestinal metaplasia and intraepithelial neoplasia developed in 3 EH monkeys but in no other groups. Transcriptional analysis of biopsies at 5 years revealed group-specific expression profiles, with striking changes in EH monkeys, plus a neoplasia-specific expression profile characterized by changes in multiple cancer-associated genes. Importantly, this neoplastic profile was evident in nonneoplastic mucosa, suggesting that the identified genes may represent markers preceding cancer. CONCLUSIONS: Gastric intraglandular neoplasia is induced in primates when H pylori infection is associated with consumption of a carcinogen similar to the nitrosaminesfound in pickled vegetables, suggesting that H pylori and the carcinogen synergistically induce gastric neoplasia in primates. Monkey arrays used for analysis of the affect of Helicobacter pylori and diet on development of gastric cancer. Each RNA was hybridized against a common reference. Biopsy numbers represent the following in terms of descriptions used in publication of the paper... Each set of numbers/descriptions is organized in the following order: Biopsy#; Monkey#; Group Code on Fig 5 Biopsy #4674; AB24; E; E1 Biopsy #4684; 16G; E; E2 Biopsy #4716; 89G; H; H1 Biopsy #4724; 54H; H; H2 Biopsy #4720; 20G; H; H3 Biopsy #4710; 92F; C; C1 Biopsy #4714; 26G; C; C2 Biopsy #4712; 92G; C; C3 Biopsy #4726; 08G; H; H4 Biopsy #4722; 57G; H; H5 Biopsy #4718; 85G; H; H6 Biopsy #4694; 48H; HE NON; HE1 Biopsy #4686; 59H; HE NON; HE2 Biopsy #4690; 81G; HE NEO; HE3 Biopsy #4696; 36G; HE NEO; HE4 Biopsy #4692; 63G; HE NEO; HE5 Biopsy #4680; 76G; E; E3 Biopsy #4682; 52G; E; E4 Biopsy #4678; 15G; E; E5 Biopsy #4676; 93G; E; E6 Biopsy #4688; 47H; HE NON; HE6 A pathogenicity experiment design type is where an infective agent such as a bacterium, virus, protozoan, fungus etc. infects a host organism(s) and the infective agent is assayed. Infection: Rhesus monkeys were infected with H. pylori (+/-) Compound Based Treatment: Rhesus monkeys were treated with ENNG (N-ethyl-N-nitrosoguanidine) (+/-) pathogenicity_design

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In situ-synthesized novel microarray optimized for mouse stem cell and early developmental expression profiling: Part 3

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In situ-synthesized novel microarray optimized for mouse stem cell and early developmental expression profiling.

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