Detection of miRNA in Cell Cultures by Using Microchip Electrophoresis with a Fluorescence-Labeled Riboprobe.docVIP

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Detection of miRNA in Cell Cultures by Using Microchip Electrophoresis with a Fluorescence-Labeled Riboprobe.doc

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Detection of miRNA in Cell Cultures by Using Microchip Electrophoresis with a Fluorescence-Labeled Riboprobe

Sensors 2012, 12, 7576-7586; doi:10.3390/s120607576 OPEN ACCESS sensors ISSN 1424-8220 /journal/sensors Article Detection of miRNA in Cell Cultures by Using Microchip Electrophoresis with a Fluorescence-Labeled Riboprobe Shohei Yamamura 1, Shouki Yatsushiro 1, Yuka Yamaguchi 1, Kaori Abe 1, Yasuo Shinohara 2 and Masatoshi Kataoka 1,* 1 Health Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 2217-14 Hayashi-cho, Takamatsu, Kagawa 761-0395, Japan; E-Mails: yamamura-s@aist.go.jp (S.Y.); yatsushiro.s@aist.go.jp (S.Y.); yukayama@suzuka-u.ac.jp (Y.Y.); k-abe@aist.go.jp (K.A.) 2 Division of Protein Expression, Institute for Genome Research, University of Tokushima, Kuramoto 3-18-15, Tokushima 770-8503, Japan; E-Mail: yshinoha@genome.tokushima-u.ac.jp * Author to whom correspondence should be addressed; E-Mail: m-kataoka@aist.go.jp; Tel.: +81-87-869-3576; Fax: +81-87-869-3551. Received: 28 March 2012; in revised form: 28 May 2012 / Accepted: 31 May 2012 / Published: 7 June 2012 Abstract: The analysis of a microRNA (miRNA), miR-222 isolated from the PC12 cell line, was performed by use of the ribonuclease (RNase) protection assay, cyanine 5 (Cy5)-labeled miR-222 riboprobe, and a Hitachi SV1210 microchip electrophoresis system, which can be used to evaluate the integrity of total RNA. The fluorescence intensity corresponding to the protected RNA fragment increased in a dose-dependent manner with respect to the complementary-strand RNA. More highly sensitive detection of miRNA by microchip electrophoresis than by conventional method using fluorescence-labeled riboprobe could be obtained in 180 s. An obvious increase in miR-222 expression induced by nerve growth factor in PC12 cells could be observed. These results clearly indicate the potential of microchip electrophoresis for the analysis of miRNA using RNase protection assay with a fluorescence-labeled riboprobe.