Nucleic acid-based diagnostics are highly sensitive and specific, but are easily disrupted by the presence of interferents in biological samples. In a laboratory or hospital setting, the influence of these interferents can be minimized using an RNA or DNA extraction procedure prior to analysis. However, in low-resource settings, limited access to specialized instrumentation and trained personnel presents challenges that impede sample preparation. We have developed a self-contained nucleic acid extraction cassette suitable for operation in a low-resource setting. This simple design contains processing solutions preloaded within a continuous length of 1.6 mm inner diameter Tygon tubing. Processing solutions are separated by air gaps and held in place during processing by the surface tension forces at the liquid–air interface, viz. surface tension valves. Nucleic acids preferentially adsorbed to silica-coated magnetic particles are separated from sample interferents using an external magnet to transfer the nucleic acid biomarker through successive solutions to precipitate, wash and elute in the final cassette solution. The efficiency of the extraction cassette was evaluated using quantitative reverse transcriptase PCR (qRT-PCR) following extraction of respiratory syncytial virus (RSV) RNA. RNA was recovered from TE buffer or from lysates of RSV infected HEp-2 cells with 55 and 33% efficiency, respectively, of the Qiagen RNeasy kit. Recovery of RSV RNA from RSV infected HEp-2 cells was similar at 30% of the RNeasy kit. An overall limit of detection after extraction was determined to be nearly identical (97.5%) to a laboratory-based commercially available kit. These results indicate that this extraction cassette design has the potential to be an effective sample preparation device suitable for use in a low-resource setting.