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Browsing by Author "Lee, Men-Jean"
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Item ASSESSMENT OF PROCEDURAL ASPECTS AND QUALITY CONTROL IN HUMAN PLACENTAL RNA ISOLATION PROTOCOLS(Office of the Vice Chancellor for Research, 2012-04-13) Maasa, Robinah K.; Sanders, Kerry L.; Creekmore, Amy L.; Lee, Men-Jean; Reiter, Jill L.High quality RNA is of paramount importance in accurately interpreting gene expression changes in the placenta throughout pregnancy, as well as in common placental pathologies. The purpose of this study was to develop a standard operating procedure for the collection of human placental tissue and isolation of high quality RNA for pregnancy-related molecular studies. To accomplish this task, we compared several different parameters to minimize RNA degradation, including preservation (liquid nitrogen vs. RNAlater), dis-ruption (mortar/pestle vs. homogenization), and isolation (Trizol vs. RNeasy). We performed 150 RNA isolations from 30 term placentas. The overall yield was 365 ± 197 ng RNA per mg of tissue. The A260/280 ratio for all samples was 2.11 ± 0.1 (mean ± s.d.) and the RQI was 7.1 ± 1.4. No significant differences in RNA purity, yield, or quality were observed between different placental collections or RNA isolation techniques. However, poor RQI values of 2.7 to 3.3 were obtained after brief thawing of frozen placental samples. We also compared storage of RNAlater stabilized tissue at 4 de-grees or room temperature for 1 day, 7 days, and 30 days. The integrity of RNA stored at room temperature for 1 day was significantly better (P‹0.05 RQI 7.3 ± 0.58, mean ± s.d) than RNA stored at room temperature for 30 days (RQI 5.0 ±1.2, mean ± s.d). The results of these studies will be useful for establishing standard procedures for placenta collection for pregnancy biobanks.Item Cytogenetic features of human trophoblast cell lines SWAN-71 and 3A-subE(Elsevier, 2017-04) Reiter, Jill L.; Drendel, Holli M.; Chakraborty, Sujata; Schellinger, Megan M.; Lee, Men-Jean; Mor, Gil; Department of Obstetrics and Gynecology, IU School of MedicineImmortalization of primary cells with telomerase is thought to maintain normal phenotypic properties and avoid chromosomal abnormalities and other cancer-associated changes that occur following simian virus 40 tumor antigen (SV40 Tag) induced immortalization. However, we report that the human telomerase reverse transcriptase (hTERT)-immortalized SWAN-71 trophoblast cell line has a near pentaploid 103∼119,XXXX[cp20] karyotype. Additionally, DNA typing analysis indicated that SWAN-71 cells have acquired microsatellite instability. In comparison, the post-crisis SV40-transformed trophoblast cell line 3A-subE was hypertriploid 69∼81,XX[cp20]. Both cell lines contained multiple specific clonal rearrangements. These findings emphasize the need to monitor for genetic instability in hTERT-immortalized cells.