Complete lab-on-a-chip sample-in, answer-out systems for nucleic acid analysis would have a wide range of applications from clinical diagnostics to environmental monitoring to genetics research. A key component in such a device is the ability to extract and purify the genetic material (DNA or RNA) in a timely, easy, reliable fashion. We present here a fully automated system capable of extracting RNA at levels comparable with manual commercial extraction methods. Purified RNA samples are delivered in less than 15 minutes from sample input. The system is highly portable, requiring only power input, a connection to a controlling computer, and an optional pressure supply.
The automated system contains a polydimethylsiloxane (PDMS) microfluidic platform complete with on-chip valves, reservoir pumps, and a disposable extraction filter. Fabrication of the PDMS chip involves a modified xurographic technique for multi-level structures. Molds were created using a scotchcal vinyl tape (3M) cut on a sign plotter (Graphtec), with higher structures being cut from acrylic on a carbon dioxide laser and positioned over the tape with an adhesive. The control and operational fluidic layers are separated by a flexible silicone membrane, and the layers are bonded using a novel masked corona discharge method.
Samples and chemicals are mixed and manipulated on the PDMS chip to isolate RNA molecules via solid phase extraction on a glass fiber filter. The system is controlled by a LabView program operating a sequence of solenoid valves, which in turn actuate the on-chip valves and reservoir pumps. The system is thus programmable and adaptable to a variety of sample types.
The device was used to demonstrate successful RNA recovery at yields approaching results attained by a Qiagen commercial spin kit . Known concentrations of RNA from an RNA standard stock (Invitrogen) were used as the sample. RNA extraction results are determined using a fluorescent Ribogreen RNA quantification kit obtained from Invitrogen. Fluorescence data is obtained using a 96-well plate reader. Binding of the nucleic acid to the PDMS surfaces was minimized using various coatings and solution stabilizers. Results from two such coating tests are presented, and extraction yields are compared to the commercial system. RNA recovery yields from a sodium poly-phosphate (NaPP) coated system (35.7 ng) approached those from the commercial spin kit (40.2 ng). However, these high-yield products displayed some NaPP contamination that interfered with reverse-transcript polymerase chain reaction (RT-PCR) analysis methods. A polyvinylpyrrolidone (PVP) coating was used to produce RT-PCR compatible outputs at the cost of a slightly reduced extraction yield (15.8 ng).
The robust nature of the system was demonstrated by extracting RNA from raw biological samples (E. coli cells) extracting up to 429 ng from samples containing 70 million bacteria cells. This highly portable, fully automated system shows promise for more widespread, universal sample preparation for RNA analysis.
See more of this Group/Topical: Topical 3: 2010 Annual Meeting of the American Electrophoresis Society (AES)