Preparation of thyroid follicular cells for mRNA quantification after fluorescence-activated cell sorting.

Chisa Matsumoto, Mitsuru Ito, Hiroya Yamada, Hiroshi Yoshida, Mikio Watanabe, Yoh Hidaka, Yoshinori Iwatani, Akira Miyauch, Toru Takano

Index: Scand. J. Clin. Lab. Invest. 73(3) , 245-52, (2013)

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Abstract

We established a novel method to analyze cells collected by fluorescence-activated cell sorting (FACS) named mRNA quantification after FACS (FACS-mQ) in which cells are labeled with a fluorescence dye in a manner that minimizes RNA degradation, and then cells sorted by FACS are examined by analyzing their gene expression profile. In order to analyze cells using FACS-mQ, it is essential to prepare single-cell suspensions without RNA degradation. We found that a new tissue preservation medium, ThelioKeep™, which contains epigallocatechin-3-gallate (EGCG), was suitable for preservation of thyroid tissues. The aim of this study was to establish a cell dispersion method of thyroid follicular cells using ThelioKeep™. We compared the efficiency of cell dispersion between the two methods, the conventional cold pre-incubation method and the ThelioKeep™ method; then we determined if cells obtained by the ThelioKeep™ method were suitable for FACS-mQ analysis. We found that a larger number of cells were recovered using ThelioKeep™ than using the conventional cold pre-incubation method. Furthermore, cell viability was higher with the ThelioKeep™ method than with the cold pre-incubation method. Thyroid cells collected by this method were analyzed by FACS-mQ. A clear shift in flow cytometry analysis was observed when cells were stained with an anti-thyroglobulin or anti-thyroid transcription factor-1 antibody. After sorting, the same copy number of ACTB mRNA was detected in thyroid cells as in an anaplastic carcinoma cell line, 8305C. These findings imply that preparation of thyroid cells using the present method is suitable for FACS-mQ analysis.