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Single cell heterogeneity plays an important role in the onset and progression of a variety of disease pathologies. One of the most notable examples of the impact of heterogeneity in the complexity of a disease is cancer. Traditionally, molecular analyses on cancer-related samples have been performed on bulk populations

Single cell heterogeneity plays an important role in the onset and progression of a variety of disease pathologies. One of the most notable examples of the impact of heterogeneity in the complexity of a disease is cancer. Traditionally, molecular analyses on cancer-related samples have been performed on bulk populations of cells, with the resultant data only representative of an average of the population, thereby concealing potentially relevant information about individual cells. Performing these studies at the single cell level is proposed to address this issue. However, current methods for the isolation and analysis of single cells often require specialized and expensive equipment that may be prohibitive to labs wishing to perform such analyses. Herein, a method for the isolation and gene expression analysis of single cells is described that (1) relies only on readily available, inexpensive materials, (2) is compatible with phase and fluorescent microscopy, and (3) allows for the ability to track specific cells throughout all measurements. This method utilizes random seeding of single cells on 72-well Terasaki plates (also called microtest plates) that have 20 µl, optically clear flat-bottomed wells in order to circumvent the need for specific hardware for cell isolation. Suspensions of the Barrett’s esophagus epithelial cell line CP-D stably expressing turboGFP and a related, GFP-negative BE cell line, CP-A, were prepared, seeded at a concentration of approximately 1-2 cells/well and incubated overnight. Wells containing single cells were visually identified using phase-contrast and fluorescent microscopy. Single cells were then lysed directly in the well, total RNA was isolated, and RT-qPCR was performed. RT-qPCR results reflected the ability to distinguish between turboGFP-expressing and non-expressing cells that matched previous identification by microscopy. These results indicate that this is a convenient and cost-effective method for studying gene expression in single cells.
ContributorsZiegler, Colleen Patricia (Author) / Chao, Joseph (Thesis director) / Tran, Thai (Committee member) / Yaron, Jordan (Committee member) / Barrett, The Honors College (Contributor) / School of Life Sciences (Contributor)
Created2013-05
Description

The microbiome and virome are known to interact within the human body which in turn modulates the health and disease of an individual. While these interactions have been largely studied in bodily sites such as the gastrointestinal tract, the microbiome and virome of the female genital tract (FGT) remains largely

The microbiome and virome are known to interact within the human body which in turn modulates the health and disease of an individual. While these interactions have been largely studied in bodily sites such as the gastrointestinal tract, the microbiome and virome of the female genital tract (FGT) remains largely understudied. Within the virome exists DNA and RNA viruses which are known to infect both eukaryotes and prokaryotes. While existing virome research within the FGT has focused largely on eukaryote infecting viruses, a large proportion of the virome consists of uncharacterized bacteriophages known as “dark matter”. Due to the lack of a specific gene marker for viruses, which is essential in qPCR quantification of other populations such as bacteria, determination of viral abundance and virome characterization has been limited. However, the staining of viral DNA has been found effective in visualizing and enumerating virus-like particles within various specimens. In this study, we seek to determine viral abundance within the FGT utilizing SYBR Gold nucleic acid stain to visualize VLP present within a cohort of cervicovaginal lavage (CVL) samples. Given these results we intend to draw conclusions regarding the interactions between the FGT virome and viral abundance as well as sexual-reproductive health. Understanding the complex relationship of the virome within the female reproductive tract is likely to have remarkable clinical implications and has the potential to progress both the diagnostic and treatment aspects of female sexual and reproductive health.

ContributorsFredenberg, Mara (Author) / Lim, Efrem (Thesis director) / Kaelin, Emily (Committee member) / Barrett, The Honors College (Contributor) / School of Life Sciences (Contributor) / School of International Letters and Cultures (Contributor)
Created2023-05