Abstract
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.
Similar content being viewed by others
References
J.D. Adams, U. Kim, H.T. Soh, Multitarget magnetic activated cell sorter. Proc. Natl. Acad. Sci. U. S. A. 105, 18165–18170 (2008)
S. Burchill, M. Bradbury, K. Pittman, J. Southgate, B. Smith, P. Selby, Detection of epithelial cancer cells in peripheral blood by reverse transcriptase-polymerase chain reaction. Br. J. Cancer 71, 278–281 (1995)
P. Chomczynski, N. Sacchi, The single-step method of RNA isolation by acid guanidinium thiocyanate–phenol–chloroform extraction: twenty-something years on. Nat. Protoc. 1, 581–585 (2006)
C.J. Easley, J.M. Karlinsey, J.M. Bienvenue, L.A. Legendre, M.G. Roper, S.H. Feldman, M.A. Hughes, E.L. Hewlett, T.J. Merkel, J.P. Ferrance, J.P. Landers, A fully integrated microfluidic genetic analysis system with sample-in–answer-out capability. Proc. Natl. Acad. Sci. U. S. A. 103, 19273–19277 (2006)
K.A. Hagan, W.L. Meier, J.P. Ferrance, J.P. Landers, Chitosan-coated silica as a solid phase for RNA purification in a microfluidic device. Anal. Chem. 81, 5249–5256 (2009)
K.A. Hagan, C.R. Reedy, M.L. Uchimoto, D. Basu, D.A. Engel, J.P. Landers, An integrated, valveless system for microfluidic purification and reverse transcription-PCR amplification of RNA for detection of infectious agents. Lab Chip 11, 957–961 (2011)
K.-H. Han, R.D. McConnell, C.J. Easley, J.M. Bienvenue, J.P. Ferrance, J.P. Landers, A.B. Frazier, An active microfluidic system packaging technology. Sensor Actuator B 122, 337–346 (2007)
J.W. Hong, V. Studer, G. Hang, W.F. Anderson, S.R. Quake, A nanoliter-scale nucleic acid processor with parallel architecture. Nat. Biotechnol. 22, 435–439 (2004)
D.W. Inglis, R. Riehn, R.H. Austin, J.C. Sturm, Continuous microfluidic immunomagnetic cell separation. Appl. Phys. Lett. 85, 5093–5095 (2004)
J. Juusola, J. Ballantyne, Messenger RNA profiling: a prototype method to supplant conventional methods for body fluid identification. Forensic Sci. Int. 135, 85–96 (2003)
H. Lee, J. Jung, S.-I. Han, K.-H. Han, High-speed RNA microextraction technology using magnetic oligo-dT beads and lateral magnetophoresis. Lab on a Chip 10, 2764–2770 (2010)
C.-S. Liao, G.-B. Lee, H.-S. Liu, T.-M. Hsieh, C.-H. Luo, Miniature RT–PCR system for diagnosis of RNA-based viruses. Nucleic Acids Res. 33, e156 (2005)
K.-Y. Lien, J.-L. Lin, C.-Y. Liu, H.-Y. Lei, G.-B. Lee, Purification and enrichment of virus samples utilizing magnetic beads on a microfluidic system. Lab Chip 7, 868–875 (2007)
J.S. Marcus, W.F. Anderson, S.R. Quake, Microfluidic single-cell mRNA isolation and analysis. Anal. Chem. 78, 3084–3089 (2006a)
J.S. Marcus, W.F. Anderson, S.R. Quake, Parallel picoliter RT-PCR assays using microfluidics. Anal. Chem. 78, 956–958 (2006b)
QIAGEN, QIAamp® RNA blood mini handbook: For total RNA purification from human whole blood, 2nd ed. (2006)
M. Setzer, J. Juusola, J. Ballantyne, Recovery and stability of RNA in vaginal swabs and blood, semen, and saliva stains. J. Forensic Sci. 53, 296–305 (2008)
T. Takano, A. Miyauchi, T. Yokozawa, F. Matsuzuka, G. Liu, T. Higashiyama, S. Morita, K. Kuma, N. Amino, Accurate and objective preoperative diagnosis of thyroid papillary carcinomas by reverse transcription-PCR detection of oncofetal fibronectin messenger RNA in fine-needle aspiration biopsies. Cancer Res. 58, 4913–4917 (1998)
Acknowledgements
This research was supported by Basic Science Research Program through the National Research Foundation of Korea funded by the Ministry of Education, Science and Technology (Grant No. 2011-0008248 to K.-H. Han) and by a grant of the National R&D Program for Cancer Control, Ministry for Health, Welfare and Family affairs, Republic of Korea (0920040 to I.-H. Choi).
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Lee, H., Han, N., Choi, IH. et al. A high-speed, high-performance on-chip integrated reverse transcription (RT)-microchip. Biomed Microdevices 15, 9–15 (2013). https://doi.org/10.1007/s10544-012-9682-3
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10544-012-9682-3