PMID- 21153374
OWN - NLM
STAT- MEDLINE
DCOM- 20110329
LR  - 20101214
IS  - 1940-6029 (Electronic)
IS  - 1064-3745 (Linking)
VI  - 680
DP  - 2011
TI  - Quantification of miRNA abundance in single cells using locked nucleic acid-FISH 
      and enzyme-labeled fluorescence.
PG  - 77-88
LID - 10.1007/978-1-60761-901-7_5 [doi]
AB  - The ability to quantify miRNA abundance at the single-cell level and image its 
      spatial distribution could lead to unique insight into the biological roles of 
      miRNAs and miRNA-associated gene regulatory networks. This protocol describes a 
      method for quantitatively imaging miRNAs in single cells using fluorescence in 
      situ hybridization (FISH). The method combines the unique miRNA recognition 
      properties of locked nucleic acid (LNA) with the signal amplification technology 
      known as enzyme-labeled fluorescence (ELF). Although both approaches have 
      previously been shown to increase detection specificity and/or sensitivity in 
      FISH, combining these techniques into one protocol allows for single molecule 
      detection. Specifically, individual miRNAs are identified as bright, photostable 
      fluorescent spots. The dynamic range was found to span over three orders of 
      magnitude and the average miRNA copy number per cell was within 17.5% of 
      measurements acquired by quantitative RT-PCR.
FAU - Lu, Jing
AU  - Lu J
AD  - Department of Bioengineering, University of Pennsylvania, Philadelphia, PA 19104, 
      USA. jingluus@yahoo.com
FAU - Tsourkas, Andrew
AU  - Tsourkas A
LA  - eng
GR  - R21-CA116102/CA/NCI NIH HHS/United States
GR  - R21-CA125088/CA/NCI NIH HHS/United States
PT  - Journal Article
PT  - Research Support, N.I.H., Extramural
PT  - Research Support, Non-U.S. Gov't
PT  - Research Support, U.S. Gov't, Non-P.H.S.
PL  - United States
TA  - Methods Mol Biol
JT  - Methods in molecular biology (Clifton, N.J.)
JID - 9214969
RN  - 0 (MicroRNAs)
RN  - 0 (Nucleic Acid Probes)
RN  - 0 (Nucleic Acids)
SB  - IM
MH  - Cells/*chemistry
MH  - Image Processing, Computer-Assisted
MH  - In Situ Hybridization, Fluorescence
MH  - MicroRNAs/*analysis
MH  - Microscopy, Fluorescence/*methods
MH  - Nucleic Acid Amplification Techniques
MH  - Nucleic Acid Probes
MH  - Nucleic Acids/*metabolism
MH  - Reverse Transcriptase Polymerase Chain Reaction
EDAT- 2010/12/15 06:00
MHDA- 2011/03/30 06:00
CRDT- 2010/12/15 06:00
PHST- 2010/12/15 06:00 [entrez]
PHST- 2010/12/15 06:00 [pubmed]
PHST- 2011/03/30 06:00 [medline]
AID - 10.1007/978-1-60761-901-7_5 [doi]
PST - ppublish
SO  - Methods Mol Biol. 2011;680:77-88. doi: 10.1007/978-1-60761-901-7_5.