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RNA sequencing of total RNA from the vagina of hormone treated C57Bl/6J mice

ABSTRACT: Purpose: The goal of this study was to compare transcriptome profiles (RNA seq) from the vaginas of uninfected mice. The mice have had ovariectomy surgeries and hormone replacement (estradiol, progesterone, vehicle, or a combination of estradiol and progesterone). Methods: 6 week old female C57Bl/6J mice had ovariectomy surgeries and a week later, started hormone replacement treatment as mentioned above. After a week of hormones, the vaginas of all mice were removed and total RNA was extracted. RNA samples were sent for RNA sequencing. Results: Upon analysis, vehicle and progesterone treated mice had similar profiles. The progesterone treated group also had progesterone stimulated genes upregulated. Estradiol regulated genes were upregulated in the estradiol treated group and the estradiol and progesterone group had a mixture of both progesterone and estradiol stimulated genes upregulated. Conclusion: This data provided a data to ensure that our hormone replacement treatments were working by the expression of certain genes based on treatment.

ORGANISM(S): Mus musculus

PROVIDER: GSE122591 | GEO | 2018/11/16

REPOSITORIES: GEO

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Project description:Despite an abundance of evidence to the contrary from animal studies, large clinical trials on humans have shown that estrogen administered to post-menopausal women increases the risk of cardiovascular disease. However, timing may be everything, as estrogen is often administered immediately after ovariectomy (ovx) in animal studies, while estrogen administration in human studies occurred many years post-menopause. This study investigates the discrepancy by administering 17ß-estradiol (E2) in a slow-release capsule to Norway Brown rats both immediately following ovx and 9 weeks post-ovx (Late), and studying differences in gene expression between these 2 groups as compared to age-matched ovx and sham operated animals. Two different types of microarray were used to analyze the left ventricles from these groups: an Affymetrix array (2 samples/group, each sample contained total RNAs pooled from 3 rats) and an Inflammatory Cytokines and Receptors PCR array (N=4 /group). Key genes were analyzed by western blotting. Ovx without replacement led to an increase in caspase 3, caspase 9, calpain 2, MMP9, and TNFα. Caspase 6, STAT3, and CD11b increased in the Late group, while TIMP2, MMP14, and collagen I α1 were decreased. MADD and fibronectin were increased in both Ovx and Late. TNFα protein levels increased with Late replacement. Many of these changes were prevented by early E2 replacement. These findings suggest that increased TNFα may be involved in some of the deleterious effects of delayed E2 administration seen in human studies. The rats were randomly assigned to four groups: Sham, Ovx, Early estrogen group, and Late estrogen group. The Sham rats were anesthetized, had their body cavities opened and then immediately closed with no tissue removal; the Ovx group underwent ovariectomy. The Early estrogen group after ovariectomy had a 0.5 mg 17-β estradiol 90-day slow release pellet (Innovative Research of America, Sarasota, FL) implanted subcutaneously in the back of the neck at the time of surgery. The Late estrogen group underwent ovariectomy and had a subcutaneous estrogen pellet placed, as above, at 9 weeks post-ovx. RNA target was prepared according to the instructions of the manufacturer (Affymetrix). For each RNA sample, good-quality total RNA from three rat hearts were pooled together in equal amounts and used to synthesize double-strand cDNA using a one-cycle cDNA synthesis kit (Affymetrix, P/N 900431). This approach allows repetitive analysis of all groups by the array in a group representative and cost effective manner, and has been used effectively by other investigators. Two pooled RNA samples were prepared for each group, and all the RNA samples were proceeded with Affymetrix expression chip analysis.

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RNA sequencing of total RNA from the vagina of hormone treated C57Bl/6J mice

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