title: Preparation of thyroid follicular cells for mRNA quantification after fluorescence-activated cell sorting.
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.
authors.affiliation: Department of Laboratory Medicine, Osaka University Graduate School of Medicine, Suita, Osaka, Japan.
authors.forename: Chisa
authors.initials: C
authors.lastname: Matsumoto
authors.forename: Mitsuru
authors.initials: M
authors.lastname: Ito
authors.forename: Hiroya
authors.initials: H
authors.lastname: Yamada
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Yoshida
authors.forename: Mikio
authors.initials: M
authors.lastname: Watanabe
authors.forename: Yoh
authors.initials: Y
authors.lastname: Hidaka
authors.forename: Yoshinori
authors.initials: Y
authors.lastname: Iwatani
authors.forename: Akira
authors.initials: A
authors.lastname: Miyauch
authors.forename: Toru
authors.initials: T
authors.lastname: Takano
mesh_terms: D000818:Animals; D000906:Antibodies / Q000737:chemistry; D002392:Catechin / Q000031:analogs & derivatives / Q000737:chemistry; D002460:Cell Line; D005434:Flow Cytometry / Q000379:methods*; D015870:Gene Expression; D006801:Humans; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D020871:RNA Stability; D012333:RNA, Messenger / Q000302:isolation & purification*; D051381:Rats; D013954:Thyroglobulin / Q000235:genetics / Q000378:metabolism; D013961:Thyroid Gland / Q000166:cytology* / Q000378:metabolism; D000074482:Thyroid Nuclear Factor 1; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism
chemical_list: D000906:Antibodies; C000614412:NKX2-1 protein, human; C000614416:Nkx2-1 protein, rat; D009687:Nuclear Proteins; D012333:RNA, Messenger; D000074482:Thyroid Nuclear Factor 1; D014157:Transcription Factors; D002392:Catechin; D013954:Thyroglobulin; C045651:epigallocatechin gallate