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High-speed RNA microextraction technology using magnetic oligo-dT beads and lateral magnetophoresis
Lee, Hwanyong,Jung, Jinhee,Han, Song-I,Han, Ki-Ho Royal Society of Chemistry 2010 Lab on a chip Vol.10 No.20
<P>This paper presents a high-speed RNA microextractor for the direct isolation of RNA from peripheral blood lysate using magnetic oligo-dT beads. The extraction is achieved through lateral magnetophoresis, generated by a ferromagnetic wire array inlaid on a glass substrate. This RNA microextractor separated more than 80% of magnetic beads with a flow rate up to 20 ml h<SUP>−1</SUP>, and the overall extraction procedure was completed within 1 min. The absorbance ratio of RNA to protein (<I>A</I><SUB>260</SUB>/<I>A</I><SUB>280</SUB>) was >1.7, indicating that the extraction technology yielded nearly pure RNA. The feasibility of this technique was evaluated further for its applicability to reverse transcription polymerase chain reaction (RT-PCR) procedures by performing cDNA synthesis and PCR. The analysis verified that the RNA microextractor is a practical method for easy, rapid, and high-precision RT-PCR using minimal reagent volumes without requiring highly trained personnel. In addition, it can be readily incorporated into genetic analysis procedures for realizing automated on-chip genetic platforms in a micro format.</P> <P>Graphic Abstract</P><P>Magnetic beads with bound RNA are laterally migrated and separated from a blood lysate, passing over an inlaid ferromagnetic wire array, by a high-speed RNA microseparator. <IMG SRC='http://pubs.rsc.org/services/images/RSCpubs.ePlatform.Service.FreeContent.ImageService.svc/ImageService/image/GA?id=c005145d'> </P>
A high-speed, high-performance on-chip integrated reverse transcription (RT)-microchip.
Lee, Hwanyong,Han, Nari,Choi, In-Hak,Han, Ki-Ho Kluwer Academic Publishers 2013 Biomedical microdevices Vol.15 No.1
<P>This report introduces an on-chip integrated reverse transcription (RT)-microchip, which includes two genetic functionalities of RNA extraction and cDNA synthesis. In the RNA extraction compartment, RNA is extracted from peripheral blood lysate within 1?min, by lateral magnetophoresis using magnetic oligo-dT beads. The extracted RNA is then collected and used directly to produce cDNA in the cDNA synthesis microchamber, which is monolithically integrated with the RNA extraction compartment. To verify the superiority of the proposed RT-microchip, RT-PCR amplification was performed using cDNA harvested from the RT-microchip, and the results were compared with those obtained using typical RNA extraction methods such as a silica matrix column and magnetic oligo-dT beads. The RT-PCR amplification results using 100?μl of blood showed that the intensity of the bands in gel electrophoresis of the RT-microchip was 2-fold stronger than that of the silica matrix column and 2.65-fold stronger than that of the magnetic oligo-dT beads. The results demonstrate that the RT-microchip technique is the most sensitive of the tested methods.</P>
올리고-dT 자성입자와 측면방향 자기영동을 이용한 초고속 RNA 추출 기술
이환용(Hwanyong Lee),한송이(Song-I Han),한기호(Ki-Ho Han) 대한기계학회 2011 大韓機械學會論文集B Vol.35 No.12
본 논문에서는 올리고-dT 자성입자와 측면방향 자기영동 기술을 기반으로 하는 초고속 RNA 추출칩을 소개한다. 센자성 와이어에 유도된 고구배자장에 의해 RNA가 결합된 올리고-dT 자성입자를 분리함으로써 용해된 혈액으로부터 고속으로 RNA를 추출하였다. 유속이 20 ㎖/h까지 자성입자를 80% 이상의 효율로 분리할 수 있었으며, 분리시간은 총 1분 이내였다. 추출된 시료로부터 단백질에 대한 RNA 흡광비율(absorbance ratio of RNA to protein: A260/A280)이 1.7 이상임을 확인하였고, 따라서 추출된 RNA가 매우 순수함을 보였다. 추출된 RNA를 사용하여 cDNA 합성과 PCR을 수행하였으며, 이로부터 개발된 초고속 RNA 추출칩이 적은 양의 시료만으로 간편하며 빠르고 정교한 RT-PCR을 수행하는데 실용적임을 확인하였다. This paper presents a high-speed RNA microextractor for the direct isolation of RNA from blood lysate using magnetic oligo(dT) beads. The extraction is performed through lateral magnetophoresis, which is induced by a ferromagnetic wire array inlaid. With this RNA microextractor, more than 80% of the magnetic beads could be separated at a flow rate up to 20 ㎖/h, and the overall extraction procedure was completed within 1 min. The absorbance ratio of RNA to protein(A260/A280) was greater than 1.7, indicating that the extraction technique yields pure RNA. The feasibility of using this technique in reverse transcription polymerase chain reaction procedures was investigated by cDNA synthesis and PCR processes. The results confirmed that the RNA microextractor is a practical device for easy, fast, and high-precision RT-PCR using minimal amounts of reagent.
이환용(Hwanyong Lee),한송이(Song-I Han),한기호(Ki-Ho Han) 대한기계학회 2010 大韓機械學會論文集B Vol.34 No.3
본 논문은 다기능 미소유체시스템의 일체형 패키징을 위한 MSI (microfluidic system interface) 기술을 제안하고, 이를 설계, 제작, 시험 평가하였다. MSI 기술을 통해 플러그 방식의 유체 인터커넥터, 유체제어를 위한 미소밸브, 광학 인터페이스를 위한 광학창을 유체시스템에 일체형으로 쉽게 구현할 수 있었다. MSI 기술의 유용성을 보이기 위해 미소 유전자시료전처리시스템에 적용되었으며, 미소 유전자시료전처리시스템은 세포정제, 세포분리, 세포용해, DNA 고체상추출, 중합효소연쇄반응, 그리고 모세관전기영동 기능으로 구성되었다. 나아가 MSI 기술이 적용된 미소 유전자시료전처리시스템의 DNA 고체상추출 및 중합효소연쇄반응의 실험결과로부터 MSI가 미소유체시스템을 위한 실용적 패키징 기술임이 검증되었다. This paper presents the technology for the design, fabrication, and characterization of a microfluidic system interface (MSI); the purpose of this technology is to enable the integration of complex microfluidic systems. The MSI technology can be applied in a simple manner for realizing complex arrangements of microfluidic interconnects, integrated microvalves for fluid control, and optical windows for on-chip optical processes. A microfluidic system for the preparation of genetic samples was used as the test vehicle to prove the effectiveness of the MSI technology for packaging complex microfluidic systems with multiple functionalities. The miniaturized genetic sample preparation system comprised several functional compartments, including compartments for cell purification, cell separation, cell lysis, solid-phase DNA extraction, polymerase chain reaction, and capillary electrophoresis. Additionally, the functional operation of the solid-phase extraction and PCR thermocycling compartments was demonstrated by using the MSI.