ZFIN ID: ZDB-PUB-160129-3
Detection and signal amplification in Zebrafish RNA FISH
Hauptmann, G., Lauter, G., Söll, I.
Date: 2016
Source: Methods (San Diego, Calif.)   98: 50-9 (Journal)
Registered Authors: Hauptmann, Giselbert, Söll, Iris
Keywords: Colocalization analysis, Gene expression profiling, Multiplex fluorescent in situ hybridization, Non-enzymatic and enzymatic signal amplification, mRNA localization
MeSH Terms:
  • Alkaline Phosphatase/chemistry
  • Animals
  • Embryo, Nonmammalian/metabolism
  • Embryo, Nonmammalian/ultrastructure*
  • Fluorescent Dyes/chemistry
  • Gene Expression Profiling
  • Gene Expression Regulation, Developmental
  • Horseradish Peroxidase/chemistry
  • In Situ Hybridization, Fluorescence/methods*
  • RNA, Messenger/chemistry*
  • RNA, Messenger/genetics
  • RNA, Messenger/metabolism
  • Signal-To-Noise Ratio
  • Single Molecule Imaging/methods*
  • Tissue Fixation/methods
  • Transcription, Genetic
  • Tyramine/chemistry
  • Zebrafish/genetics*
  • Zebrafish/growth & development
  • Zebrafish/metabolism
PubMed: 26821229 Full text @ Methods
In situ hybridization (ISH) has become an invaluable tool for the detection of RNA in cells, tissues and organisms. Due to improvements in target and signal amplification and in probe design remarkable progress has been made concerning sensitivity, specificity and resolution of chromogenic and fluorescent ISH (FISH). These advancements allow for exquisite cellular and sub-cellular resolution and for detecting multiple RNA species at a time by multiplexing. In zebrafish (F)ISH non-enzymatic and enzymatic amplification systems have been employed to obtain enhanced signal intensities and signal-to-noise ratios. These amplification strategies include branched DNA-based RNAscope and in situ hybridization chain reaction (HCR) techniques, as well as alkaline phosphatase (AP)- and horseradish peroxidase (PO)-based immunoassays. For practical application, we provide proven multiplex FISH protocols for AP- and PO-based visualization of mRNAs at high resolution. The protocols take advantage of optimized tyramide signal amplification (TSA) conditions of the PO assay and long-lasting high signal-to-noise ratio of the AP reaction, thereby enabling detection of less abundant transcripts.