MRNA quantification after fluorescence activated cell sorting using locked nucleic acid probes

Rie Maruo, Hiroya Yamada, Mikio Watanabe, Yoh Hidaka, Yoshinori Iwatani, Toru Takano

Research output: Contribution to journalArticle

9 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)42-47
Number of pages6
JournalMolecular Biotechnology
Volume49
Issue number1
DOIs
Publication statusPublished - 01-09-2011

Fingerprint

Nucleic Acid Probes
Nucleic acids
Sorting
Flow Cytometry
RNA
Fluorescence
Cells
Gene expression
Transcriptome
Oligonucleotides
Nucleic Acid Hybridization
Rats
Coloring Agents
RNA Stability
Dyes
locked nucleic acid
In Situ Hybridization
Degradation
Messenger RNA
Gene Expression

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Biotechnology
  • Molecular Biology
  • Bioengineering
  • Applied Microbiology and Biotechnology

Cite this

Maruo, Rie ; Yamada, Hiroya ; Watanabe, Mikio ; Hidaka, Yoh ; Iwatani, Yoshinori ; Takano, Toru. / MRNA quantification after fluorescence activated cell sorting using locked nucleic acid probes. In: Molecular Biotechnology. 2011 ; Vol. 49, No. 1. pp. 42-47.
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MRNA quantification after fluorescence activated cell sorting using locked nucleic acid probes. / Maruo, Rie; Yamada, Hiroya; Watanabe, Mikio; Hidaka, Yoh; Iwatani, Yoshinori; Takano, Toru.

In: Molecular Biotechnology, Vol. 49, No. 1, 01.09.2011, p. 42-47.

Research output: Contribution to journalArticle

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