PMID- 32713535
OWN - NLM
STAT- MEDLINE
DCOM- 20210623
LR  - 20210623
IS  - 1557-7988 (Electronic)
IS  - 0076-6879 (Linking)
VI  - 641
DP  - 2020
TI  - Click chemistry-based amplification and detection of endogenous RNA and DNA 
      molecules in situ using clampFISH probes.
PG  - 459-476
LID - S0076-6879(20)30177-4 [pii]
LID - 10.1016/bs.mie.2020.04.049 [doi]
AB  - Direct labeling and measurement of gene expression in single cells show the 
      tremendous variability otherwise hidden in bulk measurements. Single-molecule RNA 
      fluorescence in situ hybridization (FISH) has become a mainstay in laboratories 
      worldwide for measuring gene expression with precision. However, this method 
      remains relatively low throughput because the total fluorescent signal produced 
      is weak and requires long exposure times and high magnification microscopy, which 
      limits the total number of cells sampled in each image. As such, it is 
      experimentally difficult and time-consuming to sample a large enough population 
      of cells to visualize and quantify specific gene expression of rare cells 
      directly. Several FISH-based tools were recently developed that retain 
      single-molecule sensitivity and specificity while greatly amplifying the 
      fluorescent signal, thus making FISH-based analysis possible using standard 
      microscopes with low magnification objectives. These tools have also enabled the 
      detection of smaller and more specific targets like splice junctions or single 
      nucleotide polymorphisms. Here we will describe one such tool, clampFISH, an 
      oligonucleotide-based fluorescence amplification strategy for visualizing genomic 
      loci and individual RNA transcripts in fixed cells. ClampFISH maintains 
      specificity while amplifying fluorescent signals, making it amenable to high 
      throughput assays such as low magnification microscopy, spatial transcriptomics, 
      and flow sorting. The clampFISH technique involves probing the target RNA or DNA 
      using a series of C-shaped oligonucleotide probes, each with a 3' azide and a 5' 
      alkyne. Hybridization of the probe with the target nucleic acid brings the azide 
      and the alkyne in close proximity, allowing for ligation via bioorthogonal click 
      chemistry (CuAAC). As a result, the probe forms a closed loop around the target 
      sequence, thus enabling stringent washes to remove nonspecific binding in further 
      rounds of amplification and retention of signal throughout liquid handling steps. 
      Iterative rounds of hybridization with C-shaped, fluorescently labeled probes 
      exponentially amplify the fluorescent signal. ClampFISH is simple to implement 
      and expands the utility of in situ hybridization for multiple high throughput 
      techniques such as low magnification microscopy, flow cytometry, and sorting 
      based on RNA expression levels.
CI  - (c) 2020 Elsevier Inc. All rights reserved.
FAU - Tavakoli, Sepideh
AU  - Tavakoli S
AD  - Department of Bioengineering, Northeastern University, Boston, MA, United States.
FAU - Liu, Yifang
AU  - Liu Y
AD  - Department of Bioengineering, Northeastern University, Boston, MA, United States.
FAU - Potts, Jacob L
AU  - Potts JL
AD  - Department of Bioengineering, Northeastern University, Boston, MA, United States.
FAU - Rouhanifard, Sara H
AU  - Rouhanifard SH
AD  - Department of Bioengineering, Northeastern University, Boston, MA, United States. 
      Electronic address: s.rouhanifard@northeastern.edu.
LA  - eng
PT  - Journal Article
DEP - 20200615
PL  - United States
TA  - Methods Enzymol
JT  - Methods in enzymology
JID - 0212271
RN  - 0 (DNA Probes)
RN  - 0 (Oligonucleotide Probes)
RN  - 63231-63-0 (RNA)
RN  - 9007-49-2 (DNA)
SB  - IM
MH  - *Click Chemistry
MH  - DNA
MH  - DNA Probes/genetics
MH  - In Situ Hybridization, Fluorescence
MH  - Oligonucleotide Probes
MH  - *RNA/genetics
OTO - NOTNLM
OT  - Click chemistry
OT  - CuAAC
OT  - DNA
OT  - FISH
OT  - Fluorescence in situ hybridization
OT  - RNA
OT  - Signal amplification
OT  - Spatial transcriptome
OT  - clampFISH
EDAT- 2020/07/28 06:00
MHDA- 2021/06/24 06:00
CRDT- 2020/07/28 06:00
PHST- 2020/07/28 06:00 [entrez]
PHST- 2020/07/28 06:00 [pubmed]
PHST- 2021/06/24 06:00 [medline]
AID - S0076-6879(20)30177-4 [pii]
AID - 10.1016/bs.mie.2020.04.049 [doi]
PST - ppublish
SO  - Methods Enzymol. 2020;641:459-476. doi: 10.1016/bs.mie.2020.04.049. Epub 2020 Jun 
      15.