PMID- 32360441
OWN - NLM
STAT- MEDLINE
DCOM- 20211028
LR  - 20211028
IS  - 1095-9130 (Electronic)
IS  - 1046-2023 (Linking)
VI  - 187
DP  - 2021 Mar
TI  - Stimulated emission depletion (STED) super resolution imaging of RNA- and 
      protein-containing domains in fixed cells.
PG  - 68-76
LID - S1046-2023(19)30323-8 [pii]
LID - 10.1016/j.ymeth.2020.04.009 [doi]
AB  - Super resolution microscopy has changed our capability to visualize and 
      understand spatial arrangements of RNA- and protein-containing domains in 
      individual cells. In a previous study, we described a novel lncRNA, 
      Tumor-associated NBL2 transcript (TNBL), which originates from a primate specific 
      macrosatellite repeat. We aimed to describe several aspects of TNBL lncRNA, with 
      one focus being pinpointing its precise location in the nucleus, as well as 
      visualizing its interactions with proteins to deduce its functionality. Using a 
      combination of STimulated Emission Depletion (STED) super resolution microscopy, 
      single molecule RNA (smRNA) FISH against TNBL, and immunofluorescence against 
      SAM68 perinucleolar body, we resolved the spatial complexity of the interaction 
      between TNBL aggregates and SAM68 bodies at the perinucleolar region. Here, we 
      describe protocols for a step-by-step optimized smRNA FISH/IF and STED imaging, 
      detailing parameter settings, and three-dimensional data analysis of spatial 
      positioning of subnuclear structures. These protocols can be employed for 
      single-cell imaging of complex nuclear RNA-protein structures.
CI  - Copyright (c) 2020. Published by Elsevier Inc.
FAU - Dumbovic, Gabrijela
AU  - Dumbovic G
AD  - BioFrontiers Institute, University of Colorado at Boulder, Boulder, CO, USA. 
      Electronic address: gabrijela.dumbovic@gmail.com.
FAU - Sanjuan, Xavier
AU  - Sanjuan X
AD  - Department of Experimental and Health Sciences, Universitat Pompeu Fabra, 
      Barcelona, Spain; Advanced Light Microscopy Unit, Center for Genomic Regulation, 
      Barcelona, Spain.
FAU - Perucho, Manuel
AU  - Perucho M
AD  - Cancer Genetics and Epigenetics, Program of Predictive and Personalized Medicine 
      of Cancer (PMPPC), Health Science Research Institute Germans Trias i Pujol 
      (IGTP), Badalona, Spain; Tumor Initiation and Maintenance Program, Sanford 
      Burnham Prebys (SBP) Medical Discovery Institute, La Jolla, CA, USA.
FAU - Forcales, Sonia-V
AU  - Forcales SV
AD  - Department of Pathology and Experimental Therapeutics, Faculty of Medicine and 
      Health Sciences, University of Barcelona, L' Hospitalet de Llobregat, Barcelona, 
      Spain. Electronic address: sforcales@ub.edu.
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
DEP - 20200430
PL  - United States
TA  - Methods
JT  - Methods (San Diego, Calif.)
JID - 9426302
RN  - 0 (Adaptor Proteins, Signal Transducing)
RN  - 0 (DNA-Binding Proteins)
RN  - 0 (KHDRBS1 protein, human)
RN  - 0 (RNA, Long Noncoding)
RN  - 0 (RNA-Binding Proteins)
SB  - IM
MH  - Adaptor Proteins, Signal Transducing/*genetics
MH  - Cell Line, Tumor
MH  - Cell Nucleus/metabolism
MH  - DNA-Binding Proteins/*genetics
MH  - Epigenomics/*methods
MH  - Humans
MH  - In Situ Hybridization, Fluorescence/methods
MH  - Microscopy, Fluorescence/methods
MH  - RNA, Long Noncoding/analysis/*metabolism
MH  - RNA-Binding Proteins/*genetics
MH  - Single Molecule Imaging/*methods
MH  - Spatio-Temporal Analysis
OTO - NOTNLM
OT  - Nuclear architecture
OT  - Nuclear domains
OT  - STED
OT  - Super resolution microscopy
OT  - lncRNA
EDAT- 2020/05/04 06:00
MHDA- 2021/10/29 06:00
CRDT- 2020/05/04 06:00
PHST- 2020/02/05 00:00 [received]
PHST- 2020/04/25 00:00 [revised]
PHST- 2020/04/26 00:00 [accepted]
PHST- 2020/05/04 06:00 [pubmed]
PHST- 2021/10/29 06:00 [medline]
PHST- 2020/05/04 06:00 [entrez]
AID - S1046-2023(19)30323-8 [pii]
AID - 10.1016/j.ymeth.2020.04.009 [doi]
PST - ppublish
SO  - Methods. 2021 Mar;187:68-76. doi: 10.1016/j.ymeth.2020.04.009. Epub 2020 Apr 30.