Electronic Thesis/Dissertation


Technical Optimization of Blood RNA Isolation and Processing for Coronary Artery Disease Diagnostic Deep Sequencing Open Access

Coronary artery disease is an extremely common and very often deadly form of cardiovascular disease. Current risk assessment and diagnostic measures are invasive, costly, and sometimes inaccurate. In response to these shortcomings, a study is underway at the George Washington University to set up a blood RNA diagnostic tool using gene expression identified by deep sequencing in patients with and without otherwise diagnosed coronary artery disease. As the initial part of this study, means of sample extraction and preparation were technically optimized to yield the most informative results from patient samples. Trials were run examining four components of this process to establish the most efficient methodologies. The most ideal means of RNA isolation from whole blood samples was determined to be Tempus blood RNA collection tubes and their corresponding isolation kits. For reduction of ribosomal transcripts, which interfere with sequencing information, Ribozero kit provided the most complete depletion and globin transcript depletion was deemed unnecessary, as the presence of globin mRNA did not significantly affect downstream sequencing. Treatment using DNase in solution with Turbo DNA-free was chosen as the best way to remove all DNA from RNA samples. The methodologies which were optimized here will now be carried forward into the processing of enrolled patient samples and the continuation of this project with the intention of establishing a blood RNA based diagnostic tool for patients with suspected coronary artery disease.

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