title: Ribonuclease protection assay on microchip electrophoresis.
abstract: We describe the potential of microchip electrophoresis with a Hitachi SV1210, which can be used to evaluate the integrity of total RNA, for the analysis of mRNA expression. The ribonuclease (RNase) protection assay was performed by using microchip electrophoresis with cyanine 5 (Cy5) labeled 248-base antisense RNA probe (riboprobe) encoding adipose-type fatty acid binding protein (A-FABP) as the riboprobe. The fluorescence intensity corresponding to the protected RNA fragment increased in a dose-dependent manner with respect to the complementary strand RNA. Results were obtained in 120 s, and the same amount of Cy5-labeled antisense riboprobe as used in the conventional method can be used. Furthermore, 8 times more sensitive detection of mRNA by microchip electrophoresis could be obtained. An obvious increase in the mRNA expression of A-FABP, which is known as a differentiation marker of adipocytes, occurred during the adipocyte differentiation of 3T3-L1 cells. These results clearly indicate the potential of microchip electrophoresis for the analysis of mRNA expression in cells.
authors.affiliation: Health Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 2217-14 Hayashi-cho, Takamatsu, 761-0395, Japan.
authors.forename: Yuka
authors.initials: Y
authors.lastname: Yamaguchi
authors.forename: Shouki
authors.initials: S
authors.lastname: Yatsushiro
authors.forename: Shohei
authors.initials: S
authors.lastname: Yamamura
authors.forename: Hiroko
authors.initials: H
authors.lastname: Abe
authors.forename: Kaori
authors.initials: K
authors.lastname: Abe
authors.forename: Masahiro
authors.initials: M
authors.lastname: Watanabe
authors.forename: Kazuaki
authors.initials: K
authors.lastname: Kajimoto
authors.forename: Yasuo
authors.initials: Y
authors.lastname: Shinohara
authors.forename: Yoshinobu
authors.initials: Y
authors.lastname: Baba
authors.forename: Masatoshi
authors.initials: M
authors.lastname: Kataoka
mesh_terms: D041721:3T3-L1 Cells; D000818:Animals; D002232:Carbocyanines / Q000737:chemistry; D046209:Electrophoresis, Microchip / Q000379:methods*; D050556:Fatty Acid-Binding Proteins / Q000235:genetics / Q000378:metabolism; D008751:Methylene Blue / Q000737:chemistry; D051379:Mice; D023282:Nuclease Protection Assays / Q000379:methods*; D015347:RNA Probes / Q000737:chemistry; D016372:RNA, Antisense / Q000737:chemistry; D012333:RNA, Messenger / Q000032:analysis*; D012260:Ribonucleases / Q000378:metabolism*
chemical_list: D002232:Carbocyanines; D050556:Fatty Acid-Binding Proteins; D015347:RNA Probes; D016372:RNA, Antisense; D012333:RNA, Messenger; C085321:cyanine dye 5; D012260:Ribonucleases; D008751:Methylene Blue