YorkSpace has migrated to a new version of its software. Access our Help Resources to learn how to use the refreshed site. Contact diginit@yorku.ca if you have any questions about the migration.
 

Direct Quantitative Analysis of Multiple Micrornas

Simple item page
dc.contributor.advisorKrylov, Sergey N.
dc.creatorWegman, David Willem
dc.date.accessioned2016-09-20T18:57:07Z
dc.date.available2016-09-20T18:57:07Z
dc.date.copyright2016-05-03
dc.date.issued2016-09-20
dc.date.updated2016-09-20T18:57:07Z
dc.degree.disciplineBiology
dc.degree.levelDoctoral
dc.degree.namePhD - Doctor of Philosophy
dc.description.abstractMicroRNAs (miRNAs) play a significant role in gene regulation and have been shown to be deregulated in various diseases. Specific sets of deregulated miRNAs, termed miRNA fingerprints, can distinguish diseased from healthy samples. Detecting disease-specific fingerprints could be used in diagnostics, and there are significant efforts toward developing miRNA-detection methods for this purpose. In my project, I developed the first direct quantitative analysis of multiple miRNAs (DQAMmiR) which does not require any modifications to the target miRNAs. DQAMmiR is a hybridization assay which utilizes the separative abilities of capillary electrophoresis to analyze multiple miRNAs. I used two separation-enhancement approaches: 1) drag tags on the DNA probes to separate multiple hybrids and 2) single-strand DNA binding protein (SSB) in the run buffer to separate excess, unbound DNA probes from the hybrids. In the proof-of-principle work, I detected three miRNAs directly from a cell lysate with a limit of detection of 100 pM. I was able to further increase the number of detectable miRNAs by conjugating short peptides of varying length to the DNA probes. These peptides acted as drag tags which allowed for the separation and detection of 5 miRNAs. To lower the limit of detection I combined DQAMmiR with isotachophoresis, an in-capillary pre-concentration technique. The limit of detection improved by two orders of magnitude (from 100 pM down to 1 pM), allowing the detection of low abundance miRNAs. To improve the specificity of DQAMmiR I incorporated locked nucleic acid (LNA) bases into the probes to normalize the melting temperature of all target miRNA hybrids. This allowed me to use a single hybridization temperature, at which all target miRNA hybrids remained intact while single-nucleotide mismatches melted. Also, a dual capillary temperature technique was developed in which separation started with a high capillary temperature, required for proper hybridization, and continued at a low capillary temperature required for quality electrophoretic separation of the hybrids. I was able to combine all of these improvements to DQAMmiR while using an automated, commercially available instrument, making it an accurate, quantitative, specific, sensitive, time and cost efficient method for the analysis of multiple miRNAs.
dc.identifier.urihttp://hdl.handle.net/10315/32340
dc.language.isoen
dc.rightsAuthor owns copyright, except where explicitly noted. Please contact the author directly with licensing requests.
dc.subjectCellular biology
dc.subject.keywordsMicroRNA
dc.subject.keywordsmiRNA
dc.subject.keywordsDetection
dc.subject.keywordsCapillary electrophoresis
dc.subject.keywordsLaser induced fluorescence
dc.subject.keywordsDQAMmiR
dc.subject.keywordsIsotachophoresis
dc.subject.keywordsLocked nucleic acids
dc.subject.keywordsDiagnostic assay
dc.subject.keywordsClinical setting
dc.subject.keywordsCancer biology
dc.subject.keywordsSeparation science
dc.titleDirect Quantitative Analysis of Multiple Micrornas
dc.typeElectronic Thesis or Dissertation

Files

Original bundle
Now showing 1 - 1 of 1
Thumbnail Image
Name:
Wegman_David_W_2016_PhD.pdf
Size:
3.15 MB
Format:
Adobe Portable Document Format
Description:
Download
License bundle
Now showing 1 - 2 of 2
No Thumbnail Available
Name:
license.txt
Size:
1.83 KB
Format:
Plain Text
Description:
Download
No Thumbnail Available
Name:
YorkU_ETDlicense.txt
Size:
3.38 KB
Format:
Plain Text
Description:
Download

Collections

Biology

All items in the YorkSpace institutional repository are protected by copyright, with all rights reserved except where explicitly noted.