Chitosan-coated silica as a solid phase for RNA purification in a microfluidic device.
Chitosan-coated silica particles and chitosan-coated microchannels have been explored as an alternative to a standard silica phase for DNA extraction in a microdevice (Cao, W.; Easley, C. J.; Ferrance, J. P.; Landers, J. P. Anal. Chem. 2006, 78 (20), 7222-7228). A method that exploits the use of aqueous buffers for nucleic acid binding to and release from a solid phase is advantageous, avoiding the reagents used for conventional extraction (isopropanol and guanadinium hydrochloride), which are potent PCR inhibitors. The pH-controlled approach, which promotes nucleic acid binding to and release to the chitosan phase based on a change in buffer pH, is exploited here for RNA purification in a microfluidic device. The chitosan phase reproducibly allowed for higher RNA extraction efficiencies under aqueous conditions (71%) compared to that with a silica phase under chaotropic conditions (53%). The effectiveness of the chitosan phase was demonstrated with the successful purification of RNA from the alveolar rhabdomyosarcoma (ARMS) cancer cell line, with 3.5-fold greater extraction efficiencies than obtained when the same sample was purified using a silica phase: the resulting RNA was found to be amplifiable in reverse-transcription PCR. Low-molecular weight chitosan is also a proven inhibitor of RNases, further demonstrating the advantages of chitosan as a solid phase for RNA purification compared to silica. The chitosan phase is, therefore, a superior choice for extraction and purification of RNA in a microfluidic device and is compatible with biological samples found in a clinical or forensic setting.
Hagan
Kristin A
KA
Department of Chemistry, University of Virginia, Charlottesville, Virginia 22904, USA.
Meier
Whitney L
WL
Ferrance
Jerome P
JP
Landers
James P
JP
D045744:Cell Line, Tumor; D048271:Chitosan / Q000737:chemistry*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation* / Q000379:methods*; D012313:RNA / Q000302:isolation & purification*; D012822:Silicon Dioxide / Q000737:chemistry*; D052616:Solid Phase Extraction
D012313:RNA; D012822:Silicon Dioxide; D048271:Chitosan