Date of Award
9-25-1992
Document Type
Dissertation
Degree Name
Doctor of Arts (DA)
Department
Biology
First Advisor
William J. Wrenn
Abstract
This paper summarizes studies that were performed to evaluate the effectiveness and utility of protocols used for RNA isolation and detection. In addition, DNA probes were prepared and evaluated. Methodologies that were evaluated include the maxi-prep and mini-prep plasmid preparation techniques; isolation of RNA by extraction with organic solvents and by centrifugation through a cesium chloride gradient; and comparison of the detection of RNA-DNA hybrids using radioactively-labeled probes and digoxigenin-labeled probes. The maxi-prep and mini-prep plasmid preparation protocols both produced good quality DNA. The maxi-prep method was superior to the mini-prep method in terms of total quantity of DNA produced, but the mini-prep method was advantageous in terms of time involved in performing the procedure. RNA isolation by centrifugation through a cesium chloride gradient proved to be superior to extraction by organic solvents. RNA degradation occurred as a result of organic solvent extraction, but good quality, intact RNA was recovered after cesium chloride centrifugation. Both the radioactively-labeled and digoxigenin-labeled probes were sensitive detectors of homologous DNA sequences, and both methods were useful in detecting ribosomal RNA bands. Therefore, both of these methods should be useful in nucleic acid hybridization experiments.An investigation was also performed examining the effects of steroid hormone treatment on chick oviduct growth and total RNA production. Diethylstilbestrol (DES) administration resulted in substantial growth of the chick oviduct, whereas progesterone administration did not. Total RNA production per milligram tissue increased, although not significantly, in the oviducts of both progesterone and DES treated chicks. RNA samples from each treatment group were separated by electrophoresis on a denaturing gel to assess whether differences in RNA banding patterns could be observed, but mRNA banding was undetectable using ethidium bromide staining.
Recommended Citation
Nordlie, Margaret, "Evaluation of protocols used for RNA isolation and detection,cDNA probe labeling, and RNA quantification following hormonal stimulation of the chick oviduct." (1992). Theses and Dissertations. 7702.
https://commons.und.edu/theses/7702