mRNA quantification after fluorescence activated cell sorting using locked nucleic acid probes.
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.
Department of Laboratory Medicine, Osaka University Graduate School of Medicine, D2, 2-2 Yamadaoka, Suita, Osaka, 565-0871, Japan. marucchi_stone0611@yahoo.co.jp
Rie
R
Maruo
Hiroya
H
Yamada
Mikio
M
Watanabe
Yoh
Y
Hidaka
Yoshinori
Y
Iwatani
Toru
T
Takano
D000818:Animals; D045744:Cell Line, Tumor; D005434:Flow Cytometry / Q000379:methods*; D015870:Gene Expression; D020869:Gene Expression Profiling; D006801:Humans; D017403:In Situ Hybridization; D017404:In Situ Hybridization, Fluorescence; D015341:Nucleic Acid Probes; D020871:RNA Stability; D012333:RNA, Messenger / Q000032:analysis*; D051381:Rats; D060888:Real-Time Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D059467:Transcriptome
D015341:Nucleic Acid Probes; D012333:RNA, Messenger
Nat Protoc. 2008;3(2):190-6
18274520
RNA. 2006 Feb;12(2):187-91
16373485
Nat Protoc. 2007;2(6):1508-14
17571058
Cancer Res. 2003 Sep 15;63(18):5821-8
14522905
Cytometry A. 2009 Jan;75(1):4-13
18988270
Biochemistry. 2004 Oct 26;43(42):13233-41
15491130
Semin Cancer Biol. 2007 Jun;17(3):233-40
16569505
Semin Cancer Biol. 2008 Apr;18(2):89-102
18295505
Cancer Res. 2007 Feb 1;67(3):1030-7
17283135
Anal Biochem. 2009 Jul 15;390(2):109-14
19393610
Nature. 2008 May 15;453(7193):345-51
18480816
Nature. 2007 Jan 4;445(7123):111-5
17122771
Biochem Biophys Res Commun. 2010 Jul 2;397(3):425-8
20510885
Thyroid. 2005 May;15(5):432-8
15929664
Nature. 2001 Nov 1;414(6859):105-11
11689955
Cytometry A. 2010 Nov;77(11):1032-7
20872886
Nature. 2007 Jan 4;445(7123):106-10
17122772