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  3. Quantity and quality control of RNA isolated from foreskin, rectum, and colon samples from HIV Vaccine Trials Network 941 Trial
Title

Quantity and quality control of RNA isolated from foreskin, rectum, and colon samples from HIV Vaccine Trials Network 941 Trial

    Item Description
    Limited Access
    The author(s) chose to restrict access to this thesis to current Whitman students, faculty, and staff. Please log in to view it.
    Linked Agent
    Creator (cre): Momany, McKenzie C.
    Advisor (adv): Vernon, Dan
    Department (dpt): Whitman College. Biochemistry, Biophysics and Molecular Biology
    Date
    May 14, 2014
    Graduation Year
    2014
    Abstract

    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.

    Subject
    RNA -- Analysis -- Abnormalities
    Mucous membrane -- Diseases -- Immunological aspects -- Congresses
    Polymerase chain reaction -- Diagnostic use
    TATA-Box Binding Protein -- Isolation and purification
    Science
    Academic theses
    Whitman College 2014 -- Dissertation collection -- Biochemistry, Biophysics, and Molecular Biology
    Genre
    Theses
    Extent
    27 pages
    Permanent URL
    http://works.whitman.edu/1283
    Rights
    http://rightsstatements.org/vocab/InC/1.0/
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