Microchip-based solid-phase purification of RNA from biological samples.
Having previously detailed a method for chip-based extraction of DNA (Anal. Chem. 2003, 75, 1880-1886.), we describe here a microchip-based solid-phase extraction method for purification of RNA from biological samples is demonstrated. The method involves the use of silica beads as a solid phase, and the capacity of the device containing silica beads for RNA, RNA in the presence of protein, and DNA was determined. The capacity of the device for RNA binding in the presence of protein is 360 ng, which demonstrates sufficient capacity of the device for complete genetic analysis. An extraction of RNA can be performed on the device in as few as approximately 9 min (analytical time), a time comparable to that of a commercial extraction method, but with less reagent consumption. The microchip-based extraction is also performed in a closed system, unlike the commercial extraction method, which provides the advantage of decreased opportunity for the introduction of RNases and contaminants--essential for the sensitive RNA-based analyses presented in this work. RNA purified using the device was shown to be amplifiable using reverse transcription PCR (RT-PCR), allowing for translation of the method to the purification and subsequent amplification of biological samples. RNA was purified using the microchip-based method from neat semen, a mock semen stain, and cultured cells from a common pediatric cancer, alveolar rhabdomyosarcoma.
Department of Chemistry, University of Virginia, Charlottesville, Virginia 22904, USA.
Kristin A
KA
Hagan
Joan M
JM
Bienvenue
Christopher A
CA
Moskaluk
James P
JP
Landers
D045744:Cell Line, Tumor; D002648:Child; D006801:Humans; D046208:Microchip Analytical Procedures / Q000379:methods*; D012313:RNA / Q000302:isolation & purification*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012661:Semen / Q000737:chemistry* / Q000378:metabolism; D012822:Silicon Dioxide / Q000737:chemistry; D052616:Solid Phase Extraction / Q000379:methods*
D012333:RNA, Messenger; D012313:RNA; D012822:Silicon Dioxide