PMID- 25218386
OWN - NLM
STAT- MEDLINE
DCOM- 20150520
LR  - 20220408
IS  - 1940-6029 (Electronic)
IS  - 1064-3745 (Linking)
VI  - 1211
DP  - 2014
TI  - Simultaneous detection of nuclear and cytoplasmic RNA variants utilizing 
      Stellaris(R) RNA fluorescence in situ hybridization in adherent cells.
PG  - 189-99
LID - 10.1007/978-1-4939-1459-3_15 [doi]
AB  - RNA fluorescence in situ hybridization (FISH) has long been an indispensable tool 
      for the detection and localization of RNA and is increasingly becoming an 
      important complement to other gene expression analysis methods. We detail a 
      streamlined RNA FISH protocol for the simultaneous imaging of multiple RNA gene 
      products and RNA variants in fixed mammalian cells. The technique utilizes 
      fluorescently pre-labeled, short DNA oligonucleotides (20 nucleotides in length), 
      pooled into sets of up to 48 individual probes. The overall binding of multiple 
      oligonucleotides to the same RNA target results in punctate fluorescent signals 
      representing individual RNA molecules without the need for enzymatic signal 
      amplification. Visualization of these punctate signals, through the use of 
      wide-field fluorescence microscopy, enables the quantification of single RNA 
      transcripts. Additionally, by utilizing probe sets with spectrally distinct 
      fluorophores, multiplex analysis of specific RNAs, or RNA variants, can be 
      achieved. We focus on the detection of a cytoplasmic mRNA and a nuclear long 
      noncoding RNA to illustrate the benefits of this method for cell-specific 
      detection and subcellular localization. Post-processing of images and spot 
      counting is briefly discussed to demonstrate the capabilities of this method for 
      the statistical analysis of RNA molecule number per cell, which is information 
      that can be utilized to determine overall gene expression levels and cell-to-cell 
      gene expression variation.
FAU - Coassin, Sally R
AU  - Coassin SR
AD  - Biosearch Technologies, Inc., 2199 S. McDowell Blvd, Petaluma, CA, 94954, USA.
FAU - Orjalo, Arturo V Jr
AU  - Orjalo AV Jr
FAU - Semaan, Sheila J
AU  - Semaan SJ
FAU - Johansson, Hans E
AU  - Johansson HE
LA  - eng
PT  - Journal Article
PL  - United States
TA  - Methods Mol Biol
JT  - Methods in molecular biology (Clifton, N.J.)
JID - 9214969
RN  - 0 (Cyclin-Dependent Kinase Inhibitor p21)
RN  - 0 (MALAT1 long non-coding RNA, human)
RN  - 0 (RNA, Long Noncoding)
RN  - 0 (RNA, Messenger)
SB  - IM
MH  - Cell Adhesion
MH  - Cell Culture Techniques/methods
MH  - Cell Line, Tumor
MH  - Cell Nucleus/genetics/*ultrastructure
MH  - Cyclin-Dependent Kinase Inhibitor p21/*genetics
MH  - Cytoplasm/metabolism/*ultrastructure
MH  - Humans
MH  - In Situ Hybridization, Fluorescence/*methods
MH  - Microscopy, Fluorescence/methods
MH  - RNA, Long Noncoding/*analysis
MH  - RNA, Messenger/*analysis/genetics
EDAT- 2014/09/15 06:00
MHDA- 2015/05/21 06:00
CRDT- 2014/09/15 06:00
PHST- 2014/09/15 06:00 [entrez]
PHST- 2014/09/15 06:00 [pubmed]
PHST- 2015/05/21 06:00 [medline]
AID - 10.1007/978-1-4939-1459-3_15 [doi]
PST - ppublish
SO  - Methods Mol Biol. 2014;1211:189-99. doi: 10.1007/978-1-4939-1459-3_15.