PMID- 26721487
OWN - NLM
STAT- MEDLINE
DCOM- 20161012
LR  - 20170518
IS  - 1940-6029 (Electronic)
IS  - 1064-3745 (Linking)
VI  - 1402
DP  - 2016
TI  - Stellaris(R) RNA Fluorescence In Situ Hybridization for the Simultaneous Detection 
      of Immature and Mature Long Noncoding RNAs in Adherent Cells.
PG  - 119-134
LID - 10.1007/978-1-4939-3378-5_10 [doi]
AB  - RNA fluorescence in situ hybridization (FISH), long an indispensable tool for the 
      detection and localization of RNA, is becoming an increasingly important 
      complement to other gene expression analysis methods. Especially important for 
      long noncoding RNAs (lncRNAs), RNA FISH adds the ability to distinguish between 
      primary and mature lncRNA transcripts and thus to segregate the site of synthesis 
      from the site of action.We detail a streamlined RNA FISH protocol for the 
      simultaneous imaging of multiple primary and mature mRNA and lncRNA gene products 
      and RNA variants in fixed mammalian cells. The technique makes use of 
      fluorescently pre-labeled, short DNA oligonucleotides (circa 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 fluorescent signals 
      that reveal clusters of RNAs or single RNA molecules as punctate spots without 
      the need for enzymatic signal amplification. Visualization of these punctate 
      signals, through the use of wide-field fluorescence microscopy, enables the 
      counting of single transcripts down to one copy per cell. Additionally, by using 
      probe sets with spectrally distinct fluorophores, multiplex analysis of 
      gene-specific RNAs, or RNA variants, can be achieved. The presented examples 
      illustrate how this method can add temporospatial information between the 
      transcription event and both the location and the endurance of the mature lncRNA. 
      We also briefly discuss post-processing of images and spot counting to 
      demonstrate the capabilities of this method for the statistical analysis of RNA 
      molecules per cell. This information can be utilized to determine both overall 
      gene expression levels and cell-to-cell gene expression variation.
FAU - Orjalo, Arturo V Jr
AU  - Orjalo AV Jr
AD  - Research and Development, LGC Biosearch Technologies, 2199 S. McDowell Blvd, 
      Petaluma, CA, 94954, USA.
AD  - Biological Technologies, Analytical Development & Quality Control, Genentech, 
      Inc., 1 DNA Way, South San Francisco, CA, 94080, USA.
FAU - Johansson, Hans E
AU  - Johansson HE
AD  - Research and Development, LGC Biosearch Technologies, 2199 S. McDowell Blvd, 
      Petaluma, CA, 94954, USA. hjohansson@biosearchtech.com.
LA  - eng
PT  - Journal Article
PL  - United States
TA  - Methods Mol Biol
JT  - Methods in molecular biology (Clifton, N.J.)
JID - 9214969
RN  - 0 (RNA, Long Noncoding)
SB  - IM
MH  - Animals
MH  - Cell Adhesion
MH  - Cell Line, Tumor
MH  - Gene Expression
MH  - Humans
MH  - In Situ Hybridization, Fluorescence/*methods
MH  - Microscopy, Fluorescence
MH  - Permeability
MH  - RNA, Long Noncoding/analysis/*genetics
MH  - Tissue Fixation/methods
OTO - NOTNLM
OT  - Exon
OT  - FISH
OT  - Fluorescence
OT  - Gene expression
OT  - In situ hybridization
OT  - Intron
OT  - Nucleus
OT  - Single-molecule detection
OT  - Transcription burst
OT  - lncRNA
OT  - mRNA
OT  - qPCR
EDAT- 2016/01/02 06:00
MHDA- 2016/10/13 06:00
CRDT- 2016/01/02 06:00
PHST- 2016/01/02 06:00 [entrez]
PHST- 2016/01/02 06:00 [pubmed]
PHST- 2016/10/13 06:00 [medline]
AID - 10.1007/978-1-4939-3378-5_10 [doi]
PST - ppublish
SO  - Methods Mol Biol. 2016;1402:119-134. doi: 10.1007/978-1-4939-3378-5_10.