Circulating RNA offers a new detection approach for non-invasive diagnosis. Over the past few years, much effort has been spent on the investigation of possible detection of circulating RNA in plasma. In this thesis, we aim to quantify and characterize cell-free RNA in plasma, and investigate the possibility of using circulating fetal RNA in maternal plasma for non-invasive prenatal diagnosis or monitoring. / In the first part of the thesis, the particle-associated nature of circulating RNA was investigated. Quantitative real-time reverse-transcriptase polymerase chain reaction was developed to measure circulating RNA in healthy individuals and hepatocellular carcinoma (HCC) patients. By subjecting plasma samples to filtration and ultracentrifugation, the presence of both particle-associated and non-particle-associated mRNA species was demonstrated in human plasma. In HCC patents, both the circulating particle- and non-particle-associated plasma RNA concentrations were increased. / The discovery of circulating nucleic acids in plasma and serum has led to the development of numerous promising non-invasive diagnostic tests. The initial non-invasive tests mainly target circulating DNA species. For prenatal diagnosis, circulating fetal DNA in maternal plasma has been utilized for the non-invasive determination of a number of fetal genetic traits. However, as fetal and maternal DNA species co-exist in maternal plasma, these DNA based diagnostic applications depend largely on the use of genetic markers that could discriminate between fetal and maternal DNA, such as the Y chromosome of a male fetus. Thus, a particular DNA marker could only be used in a proportion of pregnancies. This limitation has prompted a quest to develop new fetal nucleic acid markers that are independent of sex or polymorphism and that can be used in all pregnancies. / The existence of circulating RNA is an extraordinary finding because RNA is more labile than DNA and ribonuclease is known to be present in blood. The second study of the thesis reveals that circulating RNA is surprisingly stable under different preanalytical situations. This information has made the study of circulating RNA simpler and more practical for clinical uses. / The third part of this thesis aims at detecting circulating fetal RNA in the plasma of pregnant women. We showed that two placental-derived mRNA species, namely those transcribed from the genes coding for human placental lactogen (hPL) and the beta-subunit of human chorionic gonadotropin (betahCG), are readily detectable in maternal plasma. (Abstract shortened by UMI.) / Tsui Bo Yin. / "May 2005." / Adviser: Y. M. Dennis Lo. / Source: Dissertation Abstracts International, Volume: 67-01, Section: B, page: 0074. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (p. 166-189). / 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.

Circulating RNA was previously demonstrated through the identification of tumour-derived transcripts in the plasma and serum of cancer patients. This finding inspired the detection of cell-free fetal mRNA in maternal plasma which in turn facilitated the development of promising non-invasive prenatal assessment strategies applicable to pregnancies regardless of fetal sex. / Finally, in the last study, I implemented what I have learnt from the analysis of circulating fetal RNA into the development of brain-derived RNA transcripts for detection in the plasma of patients who had sustained brain injuries. A systematic approach based on gene expression microarray analysis was adopted to search for circulating brain-specific mRNA markers. Transcripts showing high expression levels in brain tissues but low expression levels in peripheral blood were identified. However, the detectability of these brain-derived mRNA markers in both peripheral and jugular plasma was found to be low. Instead, concentrations of these mRNA markers were found to be higher in cerebral spinal fluid (CSF) from brain-injured than non-brain-injured patients. / In section IV of this thesis, I reviewed and modified the blood sample processing and plasma RNA extraction protocols that were previously practised, in an attempt to enrich circulating fetal RNA in maternal plasma. Besides mRNA, extraction protocols for microRNAs (miRNAs), a new class of circulating nucleic acid markers, were also evaluated. The modifications in the protocols that I introduced led to significant improvements in RNA yield and enhanced the accuracy and reliability of circulating RNA detection in plasma, especially for those marginally detectable transcripts. / Recently, in addition to maternal plasma, there have been studies by other research groups reporting the presence of placental/fetal mRNA in maternal whole blood. In the first part of this thesis, I studied a list of previously identified placental mRNA transcripts, including chorionic somatomammotropin hormone 1 (CSH1), KiSS-1 metastasis-suppressor (KISS1), placenta-specific 4 (PLAC4) and placenta-specific 1 (PLAC1) in maternal whole blood and determined if this whole blood-based approach offered advantages over maternal plasma analysis. The presence of KISS1, PLAC4 and PLAC1 in non-pregnant and post-partum blood samples as well as the confirmed maternal contribution of PLAC4 mRNA in maternal blood proved that most of the detected 'placental' mRNA molecules in maternal whole blood were of maternal origin. To explore if more pregnancy-associated circulating mRNA markers could be developed for maternal whole blood analysis, candidates were mined after performing gene expression microarray comparison of whole blood samples from pregnant and non-pregnant individuals. The pregnancy-specificity of the identified gene candidates was further investigated. However, their presence in non-pregnant whole blood and lack of clearance after pregnancy indicated that they were not "pregnancy-specific" markers. These data suggested that pregnancy specific transcripts could be more readily identified from maternal plasma than whole blood. / The results presented in this thesis have not only provided a foundation facilitating the precise and accurate detection of fetal-specific RNA markers but have also improved the current understanding of the biology of circulating RNA. / Heung, May Sze. / Advisers: Dennis Lo; Rossa Wk Chiu. / Source: Dissertation Abstracts International, Volume: 72-11, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 212-239). / 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, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Duzentas amostras de DNA obtidas de voluntários brasileiros não aparentados foram triadas para SNPs em uma região de 4079 pares de bases do gene PLAC4 através de reação em cadeia da polimerase (PCR) - utilizando o kit Taq Platinum DNA polymerase (Invitrogen, USA) ciclada em termociclador Eppendorf Mastercycle Gradient (Eppendorf, Germany); e posterior sequenciamento - utilizando o kit Big Dye Terminator Cycle Sequencing v3.1 (Applied Biosystems, USA) corrida em sequenciador ABI 3100 DNA Sequencer (Applied Biosystems, USA). Sete fragmentos foram amplificados utilizando pares de iniciadores desenhados com o auxílio do programa Primer 3 baseado em uma sequência do gene PLAC4 obtida do GenBank. Dez SNPs com taxa de heterozigozidade superior a 25% foram identificados, localizados em seis dos sete fragmentos estudados, que fazem a cobertura de 93% da população brasileira. Um painel combinando estes 10 SNPs apresenta potencial utilidade clínica em um teste pré-natal não invasivo da síndrome de Down fetal baseado na abordagem SNP/mRNA / Two hundred DNA samples obtained from unrelated Brazilian individuals were screened for SNPs in a region of 4079 bp of the exon of PLAC4 gene by polymerase chain reaction (PCR) - using Taq Platinum DNA polymerase kit (Invitrogen, USA) cycled on Eppendorf Mastercycle Gradient thermocycle (Eppendorf, Germany); and subsequent sequencing using Big Dye Terminator Cycle Sequencing v3.1 kit (Applied Biosystems, USA) on ABI 3100 DNA Sequencer (Applied Biosystems, USA). Seven fragments were amplified using primer pairs designed by primer 3 software based on PLAC4 sequence obtained from GenBank. Ten SNPs with heterozigosity rate above 25% were identified, located in six of the seven fragments studied, that covers up to 93% of Brazilian population. A panel combining this 10 SNPs show potential utility in clinical setting for a noninvasive prenatal diagnostic test for Down syndrome based in the SNP/mRNA approach

Duzentas amostras de DNA obtidas de voluntários brasileiros não aparentados foram triadas para SNPs em uma região de 4079 pares de bases do gene PLAC4 através de reação em cadeia da polimerase (PCR) - utilizando o kit Taq Platinum DNA polymerase (Invitrogen, USA) ciclada em termociclador Eppendorf Mastercycle Gradient (Eppendorf, Germany); e posterior sequenciamento - utilizando o kit Big Dye Terminator Cycle Sequencing v3.1 (Applied Biosystems, USA) corrida em sequenciador ABI 3100 DNA Sequencer (Applied Biosystems, USA). Sete fragmentos foram amplificados utilizando pares de iniciadores desenhados com o auxílio do programa Primer 3 baseado em uma sequência do gene PLAC4 obtida do GenBank. Dez SNPs com taxa de heterozigozidade superior a 25% foram identificados, localizados em seis dos sete fragmentos estudados, que fazem a cobertura de 93% da população brasileira. Um painel combinando estes 10 SNPs apresenta potencial utilidade clínica em um teste pré-natal não invasivo da síndrome de Down fetal baseado na abordagem SNP/mRNA / Two hundred DNA samples obtained from unrelated Brazilian individuals were screened for SNPs in a region of 4079 bp of the exon of PLAC4 gene by polymerase chain reaction (PCR) - using Taq Platinum DNA polymerase kit (Invitrogen, USA) cycled on Eppendorf Mastercycle Gradient thermocycle (Eppendorf, Germany); and subsequent sequencing using Big Dye Terminator Cycle Sequencing v3.1 kit (Applied Biosystems, USA) on ABI 3100 DNA Sequencer (Applied Biosystems, USA). Seven fragments were amplified using primer pairs designed by primer 3 software based on PLAC4 sequence obtained from GenBank. Ten SNPs with heterozigosity rate above 25% were identified, located in six of the seven fragments studied, that covers up to 93% of Brazilian population. A panel combining this 10 SNPs show potential utility in clinical setting for a noninvasive prenatal diagnostic test for Down syndrome based in the SNP/mRNA approach