Direct Quantitative Analysis of Multiple Micrornas
Wegman, David Willem
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MicroRNAs (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.