Scott M. Berry, Alex J. LaVanway, Hannah M. Pezzi, David J

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HIV Viral RNA Extraction in Wax Immiscible Filtration Assisted by Surface Tension (IFAST) Devices Scott M. Berry, Alex J. LaVanway, Hannah M. Pezzi, David J. Guckenberger, Meghan A. Anderson, Jennifer M. Loeb, David J. Beebe The Journal of Molecular Diagnostics Volume 16, Issue 3, Pages 297-304 (May 2014) DOI: 10.1016/j.jmoldx.2014.01.004 Copyright © 2014 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 1 Diagram of the IFAST design. Connected wells in the center are used for analyte extraction, whereas wells on either side are for fabrication purposes only (A); schematic of wax IFAST fabrication (B); image illustrating the difference between using a single oil barrier (left) and two oil barriers (right) (C); schematic of IFAST sample extraction procedure (D). The Journal of Molecular Diagnostics 2014 16, 297-304DOI: (10.1016/j.jmoldx.2014.01.004) Copyright © 2014 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 2 A: Correlation between sample input volume and RT-qPCR threshold cycle. Approximately 1400 VLPs were spiked into serum and loaded into each IFAST device. RNA was extracted and the level of recovered HIV RNA was measured via RT-qPCR. Error bars represent ±1 SD. B: Examples of each device size (100, 200, 350, and 500 μL). The Journal of Molecular Diagnostics 2014 16, 297-304DOI: (10.1016/j.jmoldx.2014.01.004) Copyright © 2014 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 3 Correlation between oil used and contaminant carryover. A: Error bars in the graph represent ±1 SD. B: All devices depicted in this figure were filled using silicone oil, illustrating its inability to adequately pin and separate the aqueous phases as demonstrated by the sample in the input well flooding into downstream extraction wells. The Journal of Molecular Diagnostics 2014 16, 297-304DOI: (10.1016/j.jmoldx.2014.01.004) Copyright © 2014 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 4 A: RT-qPCR measurements of RNA isolated from VLP-spiked serum samples that was prepared using IFAST and frozen immediately, raw sample stored at 37°C for 1 day, 1 week, or prepared via IFAST and then stored at 37°C for 1 week. B: Viral loads were quantified from VLP-spiked serum samples using IFAST as the RNA extraction method, allowing comparison of actual (spiked) values with the measurements. C: Viral loads were quantified from serum spiked with unmodified subtype B HIV, again using IFAST as the extraction method. Error bars represent ±1 SD. The Journal of Molecular Diagnostics 2014 16, 297-304DOI: (10.1016/j.jmoldx.2014.01.004) Copyright © 2014 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions