title: An improved protocol for mRNA quantification after fluorescence-activated cell sorting with an increased signal to noise ratio in flow cytometry.
abstract: We established a 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 fluorescent dye in a manner that minimizes RNA degradation, and then cells sorted by FACS are examined by analyzing their gene expression profile. In this study, we established a modified protocol to analyze molecules with a low expression level, such as N-cadherin and thyroid transcription factor, by improving the signal to noise ratio in flow cytometry. Use of a fluorophore-conjugated second antibody and the appropriate choice of a fluorescence dye showed a marked increase in the signal to noise ratio. Use of the Can Get Signal Immunostain in diluting antibodies shortened the reaction time. In real-time reverse transcription-PCR, a significant decrease in the copy number of intracellular mRNAs was not observed after in-tube immunostaining. These results indicated that the present protocol is useful for separating and analyzing cells by FACS-mQ, targeting a molecule with a low expression level.
authors: Department of Laboratory Medicine, Osaka University Graduate School of Medicine, D2, 2-2 Yamadaoka, Suita, Osaka, 565-0871, Japan.
authors: Arisa
authors: A
authors: Date
authors: Tomoko
authors: T
authors: Maeda
authors: Mikio
authors: M
authors: Watanabe
authors: Yoh
authors: Y
authors: Hidaka
authors: Yoshinori
authors: Y
authors: Iwatani
authors: Toru
authors: T
authors: Takano
mesh_terms: D002469:Cell Separation; D005434:Flow Cytometry; D005456:Fluorescent Dyes / Q000737:chemistry*; D006801:Humans; D020871:RNA Stability; D012333:RNA, Messenger / Q000737:chemistry / Q000302:isolation & purification*; D059629:Signal-To-Noise Ratio
chemical_list: D005456:Fluorescent Dyes; D012333:RNA, Messenger