PMID- 31524859
OWN - NLM
STAT- MEDLINE
DCOM- 20200619
LR  - 20220129
IS  - 1940-087X (Electronic)
IS  - 1940-087X (Linking)
IP  - 150
DP  - 2019 Aug 27
TI  - Quantitative Fluorescence In Situ Hybridization (FISH) and Immunofluorescence 
      (IF) of Specific Gene Products in KSHV-Infected Cells.
LID - 10.3791/59697 [doi]
AB  - Mechanistic insight arrives from careful study and quantification of specific 
      RNAs and proteins. The relative locations of these biomolecules throughout the 
      cell at specific times can be captured with fluorescence in situ hybridization 
      (FISH) and immunofluorescence (IF). During lytic herpesvirus infection, the virus 
      hijacks the host cell to preferentially express viral genes, causing changes in 
      cell morphology and behavior of biomolecules. Lytic activities are centered in 
      nuclear factories, termed viral replication compartments, which are discernable 
      only with FISH and IF. Here we describe an adaptable protocol of RNA FISH and IF 
      techniques for Kaposi's sarcoma-associated herpesvirus (KSHV)-infected cells, 
      both adherent and in suspension. The method includes steps for the development of 
      specific anti-sense oligonucleotides, double RNA FISH, RNA FISH with IF, and 
      quantitative calculations of fluorescence intensities. This protocol has been 
      successfully applied to multiple cell types, uninfected cells, latent cells, 
      lytic cells, time-courses, and cells treated with inhibitors to analyze the 
      spatiotemporal activities of specific RNAs and proteins from both the human host 
      and KSHV.
FAU - Vallery, Tenaya K
AU  - Vallery TK
AD  - Norfolk Academy.
FAU - Steitz, Joan A
AU  - Steitz JA
AD  - Department of Molecular Biophysics and Biochemistry, Howard Hughes Medical 
      Institute, Yale School of Medicine; joan.steitz@yale.edu.
LA  - eng
GR  - HHMI/Howard Hughes Medical Institute/United States
GR  - P01 CA016038/CA/NCI NIH HHS/United States
GR  - T32 AI055403/AI/NIAID NIH HHS/United States
GR  - T32 GM007223/GM/NIGMS NIH HHS/United States
PT  - Journal Article
PT  - Research Support, N.I.H., Extramural
PT  - Research Support, Non-U.S. Gov't
PT  - Video-Audio Media
DEP - 20190827
PL  - United States
TA  - J Vis Exp
JT  - Journal of visualized experiments : JoVE
JID - 101313252
RN  - 0 (Nuclear Proteins)
SB  - IM
MH  - Cell Line
MH  - Cell Nucleus/metabolism
MH  - Gene Expression Regulation, Viral
MH  - Herpesviridae Infections/*genetics
MH  - Herpesvirus 8, Human/genetics/*physiology
MH  - Humans
MH  - In Situ Hybridization, Fluorescence/*methods
MH  - Nuclear Proteins/metabolism
MH  - Virus Replication/genetics
PMC - PMC6750728
MID - NIHMS1018551
COIS- The authors have no conflicts of interest to disclose.
EDAT- 2019/09/17 06:00
MHDA- 2020/06/20 06:00
PMCR- 2020/08/27
CRDT- 2019/09/17 06:00
PHST- 2019/09/17 06:00 [entrez]
PHST- 2019/09/17 06:00 [pubmed]
PHST- 2020/06/20 06:00 [medline]
PHST- 2020/08/27 00:00 [pmc-release]
AID - 10.3791/59697 [doi]
PST - epublish
SO  - J Vis Exp. 2019 Aug 27;(150):10.3791/59697. doi: 10.3791/59697.