Visualization of Enhancer-Derived Noncoding RNA.
Enhancers are principal regulators that allow spatiotemporal tissue-specific control of gene expression. While mounting evidence suggests that enhancer-derived long noncoding RNAs (long ncRNAs), including enhancer RNAs (eRNAs), are an important component of enhancer function, their expression has not been broadly analyzed at a single cell level via imaging techniques. This protocol describes a method to image eRNA in single cells by in situ hybridization followed by tyramide signal amplification (TSA). The procedure can be multiplexed to simultaneously visualize both eRNA and protein-coding transcript at the site of transcriptional elongation, thereby permitting analysis of dynamics between the two transcript species in single cells. Our approach is not limited to eRNAs, but can be implemented on other transcripts.
Gene Expression and Biophysics Group, Synthetic Biology ERA, CSIR, Box 395, Pretoria, 0001, South Africa.|Division of Chemical Systems and Synthetic Biology, Faculty of Health Sciences, University of Cape Town, Cape Town, South Africa.
Youtaro
Y
Shibayama
Gene Expression and Biophysics Group, Synthetic Biology ERA, CSIR, Box 395, Pretoria, 0001, South Africa.|Division of Chemical Systems and Synthetic Biology, Faculty of Health Sciences, University of Cape Town, Cape Town, South Africa.
Stephanie
S
Fanucchi
Gene Expression and Biophysics Group, Synthetic Biology ERA, CSIR, Box 395, Pretoria, 0001, South Africa. musa@mhlangalab.org.|Division of Chemical Systems and Synthetic Biology, Faculty of Health Sciences, University of Cape Town, Cape Town, South Africa. musa@mhlangalab.org.|Unidade de Biofisica e Expressao Genetica, Faculdade de Medicina, Instituto de Medicina Molecular, Universidade de Lisboa, Lisbon, Portugal. musa@mhlangalab.org.
Musa M
MM
Mhlanga
D004742:Enhancer Elements, Genetic; D061307:Human Umbilical Vein Endothelial Cells; D006801:Humans; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D062085:RNA, Long Noncoding / Q000235:genetics*; D059010:Single-Cell Analysis / Q000379:methods*; D014158:Transcription, Genetic
D062085:RNA, Long Noncoding
Enhancers; Fluorescence in situ hybridization; Long ncRNA; RNA visualization; Single cell analysis; Tyramide signal amplification; eRNA