ZFIN ID: ZDB-PUB-101108-10
Programmable in situ amplification for multiplexed imaging of mRNA expression
Choi, H.M., Chang, J.Y., Trinh, L.A., Padilla, J.E., Fraser, S.E., and Pierce, N.A.
Date: 2010
Source: Nat. Biotechnol.   28(11): 1208-1212 (Journal)
Registered Authors: Fraser, Scott E., Trinh, Le
Keywords: none
MeSH Terms:
  • Animals
  • Embryo, Nonmammalian/cytology
  • Embryo, Nonmammalian/metabolism
  • Fluorescent Dyes/metabolism
  • Gene Expression Regulation
  • Humans
  • Imaging, Three-Dimensional/methods*
  • In Situ Hybridization, Fluorescence/methods*
  • Nucleic Acid Conformation
  • Polymerase Chain Reaction/methods*
  • RNA, Messenger/chemistry
  • RNA, Messenger/genetics*
  • RNA, Messenger/metabolism
  • Reproducibility of Results
  • Somites/cytology
  • Somites/metabolism
  • Tissue Fixation
  • Zebrafish/embryology
PubMed: 21037591 Full text @ Nat. Biotechnol.
In situ hybridization methods enable the mapping of mRNA expression within intact biological samples. With current approaches, it is challenging to simultaneously map multiple target mRNAs within whole-mount vertebrate embryos, representing a significant limitation in attempting to study interacting regulatory elements in systems most relevant to human development and disease. Here, we report a multiplexed fluorescent in situ hybridization method based on orthogonal amplification with hybridization chain reactions (HCR). With this approach, RNA probes complementary to mRNA targets trigger chain reactions in which fluorophore-labeled RNA hairpins self-assemble into tethered fluorescent amplification polymers. The programmability and sequence specificity of these amplification cascades enable multiple HCR amplifiers to operate orthogonally at the same time in the same sample. Robust performance is achieved when imaging five target mRNAs simultaneously in fixed whole-mount and sectioned zebrafish embryos. HCR amplifiers exhibit deep sample penetration, high signal-to-background ratios and sharp signal localization.