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mRNA Quantification After Fluorescence Activated Cell Sorting Using Locked Nucleic Acid Probes.
MedLine Citation:
PMID:  21246309     Owner:  NLM     Status:  Publisher    
Recently, we have established an in-tube in situ hybridization method named mRNA quantification after fluorescence activated cell sorting (FACS-mQ), in which a specific RNA in a particular cell type is stained with a florescent dye, allowing the stained cells to be selected by FACS without suffering excessive RNA degradation. Using this method, the biological characteristics of the sorted cells can be determined by analyzing their gene expression profile. In this study, we used locked nucleic acid (LNA) oligonucleotides, which are known to enhance both the sensitivity and specificity of RNA detection, as hybridization probes in FACS-mQ. When we used a LNA probe targeting the human 28S sequence, we were able to efficiently separate human cells from rat cells. Using LNA probes, the hybridization step was shortened to 1 h. After the hybridization step, 84.6% RNA was preserved; thus, we were able to successfully measure gene expression levels in each type of cell after FACS. Providing the LNA probe efficiently hybridizes with the target sequence, FACS-mQ with an LNA probe is a powerful tool for separating particular cells and determining their biological characteristics by analyzing their gene expression profile.
Rie Maruo; Hiroya Yamada; Mikio Watanabe; Yoh Hidaka; Yoshinori Iwatani; Toru Takano
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Publication Detail:
Type:  JOURNAL ARTICLE     Date:  2011-1-19
Journal Detail:
Title:  Molecular biotechnology     Volume:  -     ISSN:  1559-0305     ISO Abbreviation:  -     Publication Date:  2011 Jan 
Date Detail:
Created Date:  2011-1-19     Completed Date:  -     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  9423533     Medline TA:  Mol Biotechnol     Country:  -    
Other Details:
Languages:  ENG     Pagination:  -     Citation Subset:  -    
Department of Laboratory Medicine, Osaka University Graduate School of Medicine, D2, 2-2 Yamadaoka, Suita, Osaka, 565-0871, Japan,
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