title: Detection of miRNA in cell cultures by using microchip electrophoresis with a fluorescence-labeled riboprobe.
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.
authors.affiliation: Health Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Takamatsu, Kagawa, Japan. yamamura-s@aist.go.jp
authors.forename: Shohei
authors.initials: S
authors.lastname: Yamamura
authors.forename: Shouki
authors.initials: S
authors.lastname: Yatsushiro
authors.forename: Yuka
authors.initials: Y
authors.lastname: Yamaguchi
authors.forename: Kaori
authors.initials: K
authors.lastname: Abe
authors.forename: Yasuo
authors.initials: Y
authors.lastname: Shinohara
authors.forename: Masatoshi
authors.initials: M
authors.lastname: Kataoka
mesh_terms: D000818:Animals; D002232:Carbocyanines / Q000378:metabolism*; D046209:Electrophoresis, Microchip / Q000379:methods*; D004867:Equipment Design; D005456:Fluorescent Dyes / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000187:drug effects; D035683:MicroRNAs / Q000032:analysis*; D020932:Nerve Growth Factor / Q000494:pharmacology; D016716:PC12 Cells; D015347:RNA Probes / Q000378:metabolism*; D012337:RNA, Ribosomal, 18S / Q000032:analysis; D012339:RNA, Ribosomal, 28S / Q000032:analysis; D051381:Rats; D015203:Reproducibility of Results; D012260:Ribonucleases / Q000378:metabolism; D013194:Staining and Labeling
chemical_list: D002232:Carbocyanines; D005456:Fluorescent Dyes; D035683:MicroRNAs; D015347:RNA Probes; D012337:RNA, Ribosomal, 18S; D012339:RNA, Ribosomal, 28S; C085321:cyanine dye 5; D020932:Nerve Growth Factor; D012260:Ribonucleases