Finally, the fourth part of this thesis demonstrated the potential of plasma RNA integrity for noninvasive clinical diagnosis. Based on previous reports of elevated RNase activities in the circulation of cancer patients, it was hypothesized that plasma RNA integrity might serve as a useful tumor marker. Using nasopharyngeal carcinoma (NPC) as a disease model, it was found that plasma RNA in untreated NPC patients was of lower integrity than that in healthy individuals. Further analysis showed that patients undergoing radiotherapy had increased RNA integrity in the post-treatment plasma samples. These findings hence suggested that measurement of plasma RNA integrity may provide a feasible approach for noninvasive cancer detection. / Much recent interest has been focused on the clinical applications of cell-free circulating RNA for molecular diagnostics. Despite the rapid development of plasma RNA as a potential diagnostic tool, much of the biology of these molecular species remains enigmatic. This thesis aimed to investigate the integrity of cell-free RNA molecules in plasma and to study the effects of different preanalytical factors on this new biological parameter. Moreover, the possibility that plasma RNA integrity might serve as a useful clinical marker was explored. / The first part of this thesis was to develop a quantitative method for analyzing RNA integrity in plasma. One-step real-time reverse transcriptase-polymerase chain reaction (RT-PCR) was used to amplify transcript sequences corresponding to multiple regions along a housekeeping gene, glyceraldehyde-3 phosphate dehydrogenase (GAPDH). It was demonstrated that 5' transcript fragments were predominant, when compared with those derived from the middle or 3' region, in the plasma of healthy individuals. This method was validated using two-step RT-PCR and serial dilution assays. / The potential generality of the underrepresentation of 3' mRNA fragments was further studied by using circulating placental RNA as a model system. Seven transcripts were analyzed in the plasma of pregnant women: the beta subunit of human chorionic gonadotropin (betahCG), tissue factor pathway inhibitor 2 (TFPI2), adrenomedullin (ADM), inhibin beta A subunit (INHBA), placenta-specific 1 (PLAC1), pregnancy-associated plasma protein A (PAPPA), and GAPDH. The second part of this thesis showed that for five of the seven genes, there appeared to be a greater abundance of transcript fragments arising from the 5' end than those from the 3' end in maternal plasma, and that for every gene under study, the 5'-specific assay had a higher rate of detection when compared to the 3'-specific one. Apart from biological significance, these data have implications for maximizing the sensitivity of fetal RNA detection in maternal plasma for future diagnostic use. / The results presented in this thesis not only have improved the current understanding of the biological nature of cell-free circulating RNA, but also have provided important information regarding the potential clinical utility of a new parameter, plasma RNA integrity, for the field of medical diagnostics. / To ensure accurate interpretation of the results on plasma RNA integrity, a number of preanalytical issues were investigated. In the third part of this thesis, several findings were described: (1) filtration of plasma samples did not change the observation that 5' end transcript was the predominant species; (2) time delay in the processing of plasma could lead to decreased RNA concentrations despite the lack of variation in plasma RNA integrity; and (3) repeated freezing and thawing of plasma samples, but not extracted RNA, could reduce RNA integrity significantly. / Wong Chi Kwan Blenda. / "August 2006." / Adviser: Yuk Ming Dennis Lo. / Source: Dissertation Abstracts International, Volume: 68-03, Section: B, page: 1453. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (p. 148-184). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_343878 |
| Date | January 2006 |
| Contributors | Wong, Chi Kwan Blenda., Chinese University of Hong Kong Graduate School. Division of Chemical Pathology. |
| Source Sets | The Chinese University of Hong Kong |
| Language | English, Chinese |
| Detected Language | English |
| Type | Text, theses |
| Format | electronic resource, microform, microfiche, 1 online resource (xvii, 184 p. : ill.) |
| Rights | Use of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/) |
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