Ribonuclease protection assay on microchip electrophoresis
Shouki Yatsushiro,Shohei Yamamura,Hiroko Abe,Kaori Abe,Masahiro Watanabe,Kazuaki Kajimoto,Yasuo Shinohara,Masatoshi Kataoka
Analyst Pub Date : 04/21/2011 00:00:00 , DOI:10.1039/C0AN01044H
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.

Graphical abstract: Ribonuclease protection assay on microchip electrophoresis