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Quantity and quality control of RNA isolated from foreskin, rectum, and colon samples from HIV Vaccine Trials Network 941 Trial
Momany, McKenzie C.
May 14, 2014
Biochemistry, Biophysics and Molecular Biology
HIV transmission typically occurs via sexual transmission across mucosal surfaces. In order to better understand HIV infection, it is important to know what cell types and receptors are involved at various mucosal surfaces. Recent evidence has indicated that immune activation in mucosal surfaces is a critical determinant of HIV infection. HVTN914 Trial aims to quantitate immune activation in foreskin, blood and rectosigmoid mucosa from HIV-negative, uncircumcised men who have sex with men (MSM) and who are at high risk for HIV acquisition. RNA of high quality and quantity must be isolated to quantitate rare immune transcripts present in these tissues, and thus gain a better understanding of the numbers and types of HIV target cells present in different tissues. I isolated RNA from human foreskin, colon, rectum, and blood samples and evaluated the RNA in terms of yield and quality. I then amplified TATA-box-binding-protein (TBBP), an endogenous control mRNA, to determine the effects of RNA contaminants. Results from RNA yield, contamination, and integrity analysis indicated that 91% of the samples met the required quality and quantity standards for future tests and could therefore be assessed by Reverse Transcription Polymerase Chain Reaction (RT-PCR). RT-PCR results for TBBP amplification showed that blood samples amplified more rapidly than the other tissue types, indicating that contamination present in some of the mucosal tissue RNA may hinder amplification of TBBP. Overall, this project demonstrated that the methods utilized to isolate RNA from mucosal and blood samples are very effective for producing RNA of high quantity and quality. Furthermore, future PCRs can be run to quantitate receptors for HIV transmission present in different tissues, although normalization to the endogenous control will be required. This subsequent work may give us a better understanding of the concentration and distribution of HIV target cells and receptors present in various mucosal surfaces.
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