Next Article in Journal
Proteoglycan 4 Modulates Osteogenic Smooth Muscle Cell Differentiation during Vascular Remodeling and Intimal Calcification
Next Article in Special Issue
Multidimensional Single-Nuclei RNA-Seq Reconstruction of Adipose Tissue Reveals Adipocyte Plasticity Underlying Thermogenic Response
Previous Article in Journal
How Lipids Contribute to Autophagosome Biogenesis, a Critical Process in Plant Responses to Stresses
Article

Highly Sensitive and Multiplexed In Situ RNA Profiling with Cleavable Fluorescent Tyramide

Biodesign Institute & School of Molecular Sciences, Arizona State University, Tempe, AZ 85287, USA
*
Author to whom correspondence should be addressed.
Academic Editors: David Gallego-Ortega and Fatima Valdes Mora
Cells 2021, 10(6), 1277; https://doi.org/10.3390/cells10061277
Received: 24 April 2021 / Revised: 11 May 2021 / Accepted: 18 May 2021 / Published: 21 May 2021
(This article belongs to the Special Issue Single Cell Analysis of Complex Biological Systems)
Understanding the composition, regulation, and function of complex biological systems requires tools that quantify multiple transcripts at their native cellular locations. However, the current multiplexed RNA imaging technologies are limited by their relatively low sensitivity or specificity, which hinders their applications in studying highly autofluorescent tissues, such as formalin-fixed paraffin-embedded (FFPE) tissues. To address this issue, here we develop a multiplexed in situ RNA profiling approach with a high sensitivity and specificity. In this approach, transcripts are first hybridized by target-specific oligonucleotide probes in pairs. Only when these two independent probes hybridize to the target in tandem will the subsequent signal amplification by oligonucleotide hybridization occur. Afterwards, horseradish peroxidase (HRP) is applied to further amplify the signal and stain the target with cleavable fluorescent tyramide (CFT). After imaging, the fluorophores are chemically cleaved and the hybridized probes are stripped by DNase and formamide. Through cycles of RNA staining, fluorescence imaging, signal cleavage, and probe stripping, many different RNA species can be profiled at the optical resolution. In applying this approach, we demonstrated that multiplexed in situ RNA analysis can be successfully achieved in both fixed, frozen, and FFPE tissues. View Full-Text
Keywords: transcriptomics; genomics; fluorescence in situ hybridization; FISH; transcripts transcriptomics; genomics; fluorescence in situ hybridization; FISH; transcripts
Show Figures

Figure 1

MDPI and ACS Style

Xiao, L.; Labaer, J.; Guo, J. Highly Sensitive and Multiplexed In Situ RNA Profiling with Cleavable Fluorescent Tyramide. Cells 2021, 10, 1277. https://doi.org/10.3390/cells10061277

AMA Style

Xiao L, Labaer J, Guo J. Highly Sensitive and Multiplexed In Situ RNA Profiling with Cleavable Fluorescent Tyramide. Cells. 2021; 10(6):1277. https://doi.org/10.3390/cells10061277

Chicago/Turabian Style

Xiao, Lu, Joshua Labaer, and Jia Guo. 2021. "Highly Sensitive and Multiplexed In Situ RNA Profiling with Cleavable Fluorescent Tyramide" Cells 10, no. 6: 1277. https://doi.org/10.3390/cells10061277

Find Other Styles
Note that from the first issue of 2016, MDPI journals use article numbers instead of page numbers. See further details here.

Article Access Map by Country/Region

1
Back to TopTop