Loading...
Please wait, while we are loading the content...
Method and apparatus for automated isolation of nucleic acids from small cell samples
| Content Provider | NASA Technical Reports Server (NTRS) |
|---|---|
| Author | Wang, Yi Pant, Kapil Sundaram, Shivshankar Prabhakarpandian, Balabhaskar |
| Copyright Year | 2014 |
| Description | RNA isolation is a ubiquitous need, driven by current emphasis on microarrays and miniaturization. With commercial systems requiring 100,000 to 1,000,000 cells for successful isolation, there is a growing need for a small-footprint, easy-to-use device that can harvest nucleic acids from much smaller cell samples (1,000 to 10,000 cells). The process of extraction of RNA from cell cultures is a complex, multi-step one, and requires timed, asynchronous operations with multiple reagents/buffers. An added complexity is the fragility of RNA (subject to degradation) and its reactivity to surface. A novel, microfluidics-based, integrated cartridge has been developed that can fully automate the complex process of RNA isolation (lyse, capture, and elute RNA) from small cell culture samples. On-cartridge cell lysis is achieved using either reagents or high-strength electric fields made possible by the miniaturized format. Traditionally, silica-based, porous-membrane formats have been used for RNA capture, requiring slow perfusion for effective capture. In this design, high efficiency capture/elution are achieved using a microsphere-based "microfluidized" format. Electrokinetic phenomena are harnessed to actively mix microspheres with the cell lysate and capture/elution buffer, providing important advantages in extraction efficiency, processing time, and operational flexibility. Successful RNA isolation was demonstrated using both suspension (HL-60) and adherent (BHK-21) cells. Novel features associated with this development are twofold. First, novel designs that execute needed processes with improved speed and efficiency were developed. These primarily encompass electric-field-driven lysis of cells. The configurations include electrode-containing constructs, or an "electrode-less" chip design, which is easy to fabricate and mitigates fouling at the electrode surface; and the "fluidized" extraction format based on electrokinetically assisted mixing and contacting of microbeads in a shape-optimized chamber. A secondary proprietary feature is in the particular layout integrating these components to perform the desired operation of RNA isolation. Apart from a novel functional capability, advantages of the innovation include reduced or eliminated use of toxic reagents, and operator-independent extraction of RNA. |
| File Size | 92703 |
| Page Count | 2 |
| File Format | |
| Alternate Webpage(s) | http://archive.org/details/NASA_NTRS_Archive_20140002355 |
| Archival Resource Key | ark:/13960/t0dv6j84j |
| Language | English |
| Publisher Date | 2014-01-01 |
| Access Restriction | Open |
| Subject Keyword | Electrodes Ribonucleic Acids Electrokinetics Microfluidic Devices Extraction Culture Techniques Microparticles Ntrs Nasa Technical Reports Server (ntrs) Nasa Technical Reports Server Aerodynamics Aircraft Aerospace Engineering Aerospace Aeronautic Space Science |
| Content Type | Text |
| Resource Type | Technical Report |
