In tube immunocytochemistry for fluorescence-activated cell sorting that prevents RNA degradation in sorted cells.
Biotech Histochem
; 95(1): 1-7, 2020 Jan.
Article
em En
| MEDLINE
| ID: mdl-31423857
ABSTRACT
Fluorescence-activated cell sorting (FACS) is a powerful tool for analyzing stem cells. When using fixed cells, however, it is sometimes difficult to analyze RNA extracted from sorted cells due to RNA degradation. We established a protocol for immunocytochemistry before FACS to prevent RNA degradation. Cells were fixed with a methanol-based fixative (UM-Fix), then subjected to immunocytochemistry. The addition of RNase inhibitor and dithiothreitol (DTT) to some buffers used for immunocytochemistry increased RNA integrity after cell recovery. We found increased copy numbers of mRNA in recovered cells using quantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis. When RNase inhibitor and DTT were added, amplification of mRNA using T7 promoter was possible with RNA extracted from recovered cells after FACS. Our protocol ensures high quality RNA in cells recovered by FACS; therefore, gene expression analysis with a smaller number of cells is possible using pre-amplification of mRNAs. Our protocol for immunocytochemistry also might be applicable to RNA recovery after immunostaining.
Palavras-chave
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
RNA
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Imuno-Histoquímica
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Caderinas
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Citometria de Fluxo
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Fator Nuclear 1 de Tireoide
Limite:
Animals
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Humans
Idioma:
En
Revista:
Biotech Histochem
Ano de publicação:
2020
Tipo de documento:
Article