Graduation Date


Document Type

Master's Thesis (Campus only Access)

Degree Name

Master of Science

Department or Program

Clinical Laboratory Sciences

Department or Program Chair

Mary Sevigny, PhD

First Reader

Marla Vaughn

Second Reader

Maria C. DeSousa, JD, MPA, CLS


Cell-free DNA (cfDNA) is an emerging biomarker that researchers are utilizing to detect diseases in patients, allowing physicians to accurately diagnose and treat patients at the individual level. A few applications that utilize cfDNA to detect diseases in IVD (in-vitro diagnostics) are non-invasive prenatal testing, circulating tumor DNA markers, transplant rejection using donor derived cfDNA and microbial cfDNA in identifying infectious agents (3,4,5,6). The current challenge in our laboratory is to measure cfDNA concentration from minimal volume of plasma after the extraction methods. Different methods of extraction yield various concentrations of dsDNA. Our laboratory develops the use of a modified PicoGreen kits and Biotek’s microplate reader to measure cfDNA concentration. This assay is being qualified to allow the laboratory to assess the yields of dsDNA after extraction method. Qualification includes performance for linearity, accuracy, precision and stability of standards. The study of this modified PicoGreen assay establishes the linearity assay range from 1ng/mL to 1200ng/mL (equivalent to 0.017 – 20 ng/uL of dsDNA) with the imprecision less than 20%. The accuracy study was verified using commercial material with a known concentration and a comparison method with Agilent’s Tape-station. The standards of this assay have 14 days stability. The modified PicoGreen assay’s performance metrics are evaluated and determined to be acceptable for laboratory’s purposes, to use as quality indicator in assessing the yield of dsDNA from laboratory’s extraction method.

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