Open AccessVisualization of global RNA synthesis in a human (mini-) organ in situ by click chemistry
Author(s) -
Talveen S. Purba,
John Marsh,
Kayumba Ng'andu,
Svitlana Kurinna,
Ralf Paus
Publication year - 2018
Publication title -
biotechniques/biotechniques
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.617
H-Index - 131
eISSN - 1940-9818
pISSN - 0736-6205
DOI - 10.2144/btn-2018-0025
Subject(s) - rna , in situ , ex vivo , microbiology and biotechnology , biology , click chemistry , in vivo , in situ hybridization , chemistry , protein biosynthesis , biochemistry , computational biology , messenger rna , in vitro , genetics , gene , combinatorial chemistry , organic chemistry
RNA synthesis can be detected by 5-ethynyl uridine (EU) incorporation and click chemistry. Despite identifying a fundamental functional process, this technique has yet to be widely applied to complex human tissue systems. By incorporating EU into human hair follicle (HF) organs cultured ex vivo, nascent RNA synthesis was detected in situ. EU differentially incorporated across the HF epithelium. Interestingly, RNA synthesis did not correlate with protein synthesis, proliferation or epithelial progenitor cell marker expression. By treating human HFs with the cytotoxic cell cycle inhibitor (R)-CR8, which inhibits transcriptional regulators CDK7 and CDK9, it was further shown that this technique can be used to sensitively detect changes in global RNA synthesis in situ. Together, this work delineates new insights into nascent RNA synthesis within a human (mini)- organ and describes a novel read-out parameter that will enrich future ex vivo human tissue research studies.