Establishment of the protocol for mRNA quantification after FACS.

2016 Fiscal Year Final Research Report

• Project/Area Number: 26460676
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Multi-year Fund
• Section: 一般
• Research Field: Laboratory medicine
• Research Institution: Osaka University
• Principal Investigator: Takano Toru 大阪大学, 医学系研究科, 講師 (00263236)
• Project Period (FY): 2014-04-01 – 2017-03-31
• Keywords: Flow cytometry / 癌幹細胞

Outline of Final Research Achievements

In our previous studies, we established a method to analyze cells collected by fluorescence-activated cell sorting (FACS), named mRNA quantification after FACS (FACS-mQ). In FACS-mQ, cells are labeled with a fluorescence dyes, and then cells sorted by FACS are examined by analyzing their gene expression profile. Using the previous FACS-mQ protocol, it was not possible to apply some methods, such as amplification of RNAs using T7 promoter, due to RNA degradation. In this study, we tried to modify the FACS-mQ protocol to prevent RNA degradation during FACS. Addition of RNase inhibitor and DTT to some buffers used in FACS-mQ resulted in a high RNA integrity number after FACS. Furthermore, no significant differences in the images of flow cytometry were observed between specimens with or without RNase inhibitor and DTT. Thus, this modification to the FACS-mQ protocol assured the quality of RNAs in cells recovered by FACS.

Free Research Field

臨床検査医学