Purpose To study gene expression at single embryonic stem cell colony levels with a new RT-PCR protocol. Methods Forty-five mouse ES cell colonies were retrieved at the 5th, 10th, 15th, 20th and 25th passages. The pluripotent state was analyzed for OCT-4 and Nanog, and β-actin as a control for the presence of templates. RT-PCR was done using the SuperScript™ III CellsDirect cDNA Synthesis System. Every 2 or 3 days just before passage, a single colony was loaded into a 0.5 ml PCR tube containing 10 μl of resuspension buffer using a pulled glass pipette. Results The RT-PCR protocol was completed in less then 150 min. All colonies were positive for OCT-4 and β-actin and 42 out of 45 were positive for Nanog. Conclusions This protocol requires as little as 10 pg of total RNA starting material and is therefore useful for low cell number tissues, such as single stem cell colonies or preimplantation embryonic materials.
Background. The aim of this study was to evaluate the action of tamoxifen on the endometrium in states of chronic anovulation.
Methods. Thirty‐eight rats inducted to persistent estrous (testosterone propionate) confirmed by hormonal colpocytology were divided into a control and an experimental group; the latter received tamoxifen and had fragments of the uterine horns processed for morphological and morphometrical analysis. Data were analysed statistically by the Mann‐Whitney and Student's t tests.
Results. Our findings revealed minor uterine weight, epithelial thickness; number of endometrial glands and low eosinophil counts in the group that received tamoxifen. These results were statistically significant. We often observed areas of metaplasic stratified squamous epithelium between cylindrical epithelial cells in both groups.
Conclusions. Our results indicate that antiestrogenic effect of tamoxifen was only partial in persistent estrous, since there was no blocking against the squamous metaplasia of the endometrium.
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