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Gene Expression Profiles of tumor sorted fractions demarcating genetic instability using DNA-RNA content based cell cycle analysis in ovarian cancer xenograft tumor

ABSTRACT: Our earlier report states role of genetic instability and aneuploid cells in drug resistance and tumor recurrence. Hence segregating tumor based on DNA content is a good model to study genetic in stability. We have earlier reported isolation of cell line (A4T) from a patient with Grade IV serous adenocarcinoma (Bapat et al. Cancer Research. 2005). Xenograft generated by injecting A4T cells in NOD/SCID mice exhibited genetic instability (Kusumbe and Bapat Cancer Research. 2009; Naik et.al Oncogene 2015). We generated xenograft by injecting 2.5x106 A4T P65 s.c in NOD/SCID mice. Mice were observed for tumor formation and tumors were harvested at day 28 (4 weeks) and processed for collagenase digestion for preparation of single cell suspension. Further cells were subjected to staining with Hoechst (33342) for 45min at 37˚C followed by pyronin Y for 45min at 37˚C and processed for FACS sorting. Staining profile was acquired on BD FACS Aria II across UV laser for Hoechst and PE channel for PyroninY with linear scale. Details of the Hoechst pyronin Y based ploidy and cell cycle analysis are published in our earlier reports (Kusumbe and Bapat Cancer Research. 2009; Naik et.al Oncogene 2015). DNA content of cell line injected is considered as euploid while the higher genetic content is called as aneuploid.

ORGANISM(S): Homo sapiens

PROVIDER: GSE74852 | GEO | 2016/01/01

SECONDARY ACCESSION(S): PRJNA301679

REPOSITORIES: GEO

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Gene Expression Profiles of tumor sorted fractions demarcating genetic instability using DNA-RNA content based cell cycle analysis in ovarian cancer xenograft tumor

Project description:Our earlier report states role of genetic instability and aneuploid cells in drug resistance and tumor recurrence. Hence segregating tumor based on DNA content is a good model to study genetic in stability. We have earlier reported isolation of cell line (A4T) from a patient with Grade IV serous adenocarcinoma (Bapat et al. Cancer Research. 2005). Xenograft generated by injecting A4T cells in NOD/SCID mice exhibited genetic instability (Kusumbe and Bapat Cancer Research. 2009; Naik et.al Oncogene 2015). We generated xenograft by injecting 2.5x106 A4T P65 s.c in NOD/SCID mice. Mice were observed for tumor formation and tumors were harvested at day 28 (4 weeks) and processed for collagenase digestion for preparation of single cell suspension. Further cells were subjected to staining with Hoechst (33342) for 45min at 37ËC followed by pyronin Y for 45min at 37ËC and processed for FACS sorting. Staining profile was acquired on BD FACS Aria II across UV laser for Hoechst and PE channel for PyroninY with linear scale. Details of the Hoechst pyronin Y based ploidy and cell cycle analysis are published in our earlier reports (Kusumbe and Bapat Cancer Research. 2009; Naik et.al Oncogene 2015). DNA content of cell line injected is considered as euploid while the higher genetic content is called as aneuploid. Euploid and aneuploid were sorted with 99% purity and 1x105 cells from each fraction was subjected to RNA isolation, labeling and microarray analysis. RNA of each fraction was labeled with Cy3 and designated as A4Euploid and A4Aneuploid. Three samples of each were chosen for hybridization.

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