Mass spectrometric methods and bioinformatics tools for accurate identification of MicroRNA biomarkers
- UNCG Author/Contributor (non-UNCG co-authors, if there are any, appear on document)
- Dickson M. Wambua (Creator)
- Institution
- The University of North Carolina at Greensboro (UNCG )
- Web Site: http://library.uncg.edu/
- Advisor
- Norman Chiu
Abstract: MicroRNA (miRNA) are a class of endogenous non-protein-coding RNA of ~19-25 nucleotides long that post-transcriptionally regulate protein expression by targeting messenger RNAs for cleavage or translational repression. MiRNAs have been implicated in the initiation and progression of 160+ human diseases. Unique miRNA differential expression signatures can be used as a basis of discriminating against the presence or absence of human diseases. MiRNAs are therefore a promising and emerging class of disease biomarkers and therapeutic targets; however, the accurate detection of a specific miRNA has continued to be a challenging issue. Recently, mass spectrometry (MS) has seen remarkable technological advancements making it an attractive alternative to the conventional molecular biology miRNA characterization techniques. This study consistently documents the development of various analytical techniques aimed at characterization of miRNAs. The current literature in the field of miRNA is covered in chapter one. In chapter two, two new MS based concepts for detection of miRNA are introduced; a) the miRNA is captured using a specific complementary DNA probe, eluted and digested with specific endonuclease. The digested miRNA fragments are measured by MS resulting in a peak pattern that is dependent on the miRNA sequence i.e. an intrinsic mass signature and b) a unique mass signature is created by incorporating extra nucleotide(s) to the 3' end of miRNA and the extended miRNA is measured by using MS. The molecular mass of the extended miRNA, which is defined as extended mass signature, is expected to be different from the other miRNA within the same sample. These two approaches can improve the accuracy on qualitative MS identification of specific miRNA. To better understand miRNA function however, it is important to elucidate the nucleotide sequence of the miRNA. Chapter three of this study introduces a novel MS based assay for the sequencing of miRNA through chemical hydrolysis. In this study, by taking advantage of the mixing between a miRNA sample and an acidic MALDI matrix prior to the MALDI-TOF MS measurements, a unique yet simple and relatively cost-effective approach to generate miRNA sequencing ladders was developed. By using this method, 100% sequence coverage and accuracy in the sequencing of selected miRNAs were achieved. When many samples are involved, the data generated from miRNA measurements can be complex and manual data processing is tedious and challenging, as such, the spectral interpretation of mass spectrometric data can quickly turn out to be the bottleneck in miRNA analysis. The success of MS as a tool for analysis of miRNA will therefore strongly depend on the development of relevant computational software with the ability to properly interpret and analyze the large data. To meet this need, chapter four of this work explains the development of MicroRNA MultiTool, a computational software for the rapid interpretation of MS data containing human miRNA. Users can directly enter data obtained from mass spectrometric measurement in order to obtain the identify of miRNA, highly reducing the time needed to process data. The development of such analytical and bioinformatics tools will provide scientists with the opportunity to better understand miRNA functions and will be influential in propelling the breakthroughs of miRNA in clinical diagnostics and therapeutic fields.
Mass spectrometric methods and bioinformatics tools for accurate identification of MicroRNA biomarkers
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Created on 12/1/2012
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Additional Information
- Publication
- Dissertation
- Language: English
- Date: 2012
- Keywords
- Mass Spectrometry, MicroRNA, MicroRNA Bioinformatics, MicroRNA biomarkers, MultiTool, RNA Extension
- Subjects
- Non-coding RNA
- Mass spectrometry
- Biochemical markers