title: Selective nucleic acid removal via exclusion (SNARE): capturing mRNA and DNA from a single sample.
abstract: The path from gene (DNA) to gene product (RNA or protein) is the foundation of genotype giving rise to phenotype. Comparison of genomic analyses (DNA) with paired transcriptomic studies (mRNA) is critical to evaluating the pathogenic processes that give rise to human disease. The ability to analyze both DNA and mRNA from the same sample is not only important for biologic interrogation but also to minimize variance (e.g., sample loss) unrelated to the biology. Existing methods for RNA and DNA purification from a single sample are typically time-consuming and labor intensive or require large sample sizes to split for separate RNA and DNA extraction procedures. Thus, there is a need for more efficient and cost-effective methods to purify both RNA and DNA from a single sample. To address this need, we have developed a technique, termed SNARE (Selective Nucleic Acid Removal via Exclusion), that uses pinned oil interfaces to simultaneous purify mRNA and DNA from a single sample. A unique advantage of SNARE is the elimination of dilutive wash and centrifugation processes that are fundamental to conventional methods where sample is typically discarded. This minimizes loss and maximizes recovery by allowing nondilutive reinterrogation of the sample. We demonstrate that SNARE is more sensitive than commercially available kits, robustly and repeatably achieving mRNA and DNA purification from extremely low numbers of cells for downstream analyses. In addition to sensitivity, SNARE is fast, easy to use, and cost-effective and requires no laboratory infrastructure or hazardous chemicals. We demonstrate the clinical utility of the SNARE with prostate cancer circulating tumor cells to demonstrate its ability to perform both genomic and transcriptomic interrogation on rare cell populations that would be difficult to achieve with any current method.
authors.affiliation: Department of Biomedical Engineering, Wisconsin Institutes for Medical Research, University of Wisconsin-Madison , 1111 Highland Avenue, Madison, Wisconsin53706, United States.
authors.forename: Lindsay
authors.initials: L
authors.lastname: Strotman
authors.forename: Rachel
authors.initials: R
authors.lastname: O'Connell
authors.forename: Benjamin P
authors.initials: BP
authors.lastname: Casavant
authors.forename: Scott M
authors.initials: SM
authors.lastname: Berry
authors.forename: Jamie M
authors.initials: JM
authors.lastname: Sperger
authors.forename: Joshua M
authors.initials: JM
authors.lastname: Lang
authors.forename: David J
authors.initials: DJ
authors.lastname: Beebe
mesh_terms: D001483:Base Sequence; D002021:Buffers; D045744:Cell Line, Tumor; D005591:Chemical Fractionation / Q000379:methods*; D004247:DNA / Q000235:genetics / Q000302:isolation & purification*; D006112:Gravitation; D006801:Humans; D008297:Male; D046210:Microfluidic Analytical Techniques / Q000379:methods*; D009154:Mutation; D011471:Prostatic Neoplasms / Q000235:genetics / Q000473:pathology; D012333:RNA, Messenger / Q000235:genetics / Q000302:isolation & purification; D013500:Surface Tension
chemical_list: D002021:Buffers; D012333:RNA, Messenger; D004247:DNA
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