220-plex microRNA expression profile of a single cell

被引：72

作者：

Tang, Fuchou

Hajkova, Petra

Barton, Sheila C.

O'Carroll, Donal

Lee, Caroline

Lao, Kaiqin

Surani, M. Azim

机构：

[1] Univ Cambridge, Wellcome Trust Canc Res UK Gurdon Inst Canc & Dev, Cambridge CB2 1QN, England

[2] Rockefeller Univ, Howard Hughes Med Inst, Lab Lymphocyte Signalling, New York, NY 10021 USA

[3] Rockefeller Univ, Howard Hughes Med Inst, Lab Mol Immunol, New York, NY 10021 USA

[4] Appl Biosyst Inc, Adv Res Technol, Foster City, CA 94404 USA

来源：

NATURE PROTOCOLS | 2006年 / 1卷 / 03期

基金：

英国惠康基金;

关键词：

D O I：

10.1038/nprot.2006.161

中图分类号：

Q5 [生物化学];

学科分类号：

071010 ; 081704 ;

摘要：

Here we describe a protocol for the detection of the microRNA (miRNA) expression profile of a single cell by stem-looped real-time PCR, which is specific to mature miRNAs. A single cell is first lysed by heat treatment without further purification. Then, 220 known miRNAs are reverse transcribed into corresponding cDNAs by stem-looped primers. This is followed by an initial PCR step to amplify the cDNAs and generate enough material to permit separate multiplex detection. The diluted initial PCR product is used as a template to check individual miRNA expression by real-time PCR. This sensitive technique permits miRNA expression profiling from a single cell, and allows analysis of a few cells from early embryos as well as individual cells (such as stem cells). It can also be used when only nanogram amounts of rare samples are available. The protocol can be completed in 7 d.

引用

收藏

页码：1154 / 1159

页数：6

相关论文

共 30 条

[1] MicroRNA functions in animal development and human disease [J].

Alvarez-Garcia, I ;

Miska, EA .

DEVELOPMENT, 2005, 132 (21) :4653-4662

[2] The functions of animal microRNAs [J].

Ambros, V .

NATURE, 2004, 431 (7006) :350-355

[3] Probing microRNAs with microarrays: Tissue specificity and functional inference [J].

Babak, T ;

Zhang, W ;

Morris, Q ;

Blencowe, BJ ;

Hughes, TR .

RNA, 2004, 10 (11) :1813-1819

[4] MicroRNA expression detected by oligonucleotide microarrays: System establishment and expression profiling in human tissues [J].

Barad, O ;

Meiri, E ;

Avniel, A ;

Aharonov, R ;

Barzilai, A ;

Bentwich, I ;

Einav, U ;

Glad, S ;

Hurban, P ;

Karov, Y ;

Lobenhofer, EK ;

Sharon, E ;

Shiboleth, YM ;

Shtutman, M ;

Bentwich, Z ;

Einat, P .

GENOME RESEARCH, 2004, 14 (12) :2486-2494

[5] Microarray profiling of microRNAs reveals frequent coexpression with neighboring miRNAs and host genes [J].

Baskerville, S ;

Bartel, DP .

RNA, 2005, 11 (03) :241-247

[6] MicroRNA profiling reveals distinct signatures in B cell chronic lymphocytic leukemias [J].

Calin, GA ;

Liu, CG ;

Sevignani, C ;

Ferracin, M ;

Felli, N ;

Dumitru, CD ;

Shimizu, M ;

Cimmino, A ;

Zupo, S ;

Dono, M ;

Dell'Aquila, ML ;

Alder, H ;

Rassenti, L ;

Kipps, TJ ;

Bullrich, F ;

Negrini, M ;

Croce, CM .

PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 2004, 101 (32) :11755-11760

[7] Real-time quantification of microRNAs by stem-loop RT-PCR [J].

Chen, CF ;

Ridzon, DA ;

Broomer, AJ ;

Zhou, ZH ;

Lee, DH ;

Nguyen, JT ;

Barbisin, M ;

Xu, NL ;

Mahuvakar, VR ;

Andersen, MR ;

Lao, KQ ;

Livak, KJ ;

Guegler, KJ .

NUCLEIC ACIDS RESEARCH, 2005, 33 (20) :e179.1-e179.9

[8] T cell lineage choice and differentiation in the absence of the RNase III enzyme Dicer [J].

Cobb, BS ;

Nesterova, TB ;

Thompson, E ;

Hertweck, A ;

O'Connor, E ;

Godwin, J ;

Wilson, CB ;

Brockdorff, N ;

Fisher, AG ;

Smale, ST ;

Merkenschlager, M .

JOURNAL OF EXPERIMENTAL MEDICINE, 2005, 201 (09) :1367-1373

[9] PREGNANCIES FROM BIOPSIED HUMAN PREIMPLANTATION EMBRYOS SEXED BY Y-SPECIFIC DNA AMPLIFICATION [J].

HANDYSIDE, AH ;

KONTOGIANNI, EH ;

HARDY, K ;

WINSTON, RML .

NATURE, 1990, 344 (6268) :768-770

[10] MicroRNAs in vertebrate development [J].

Harfe, BD .

CURRENT OPINION IN GENETICS & DEVELOPMENT, 2005, 15 (04) :410-415