TY - JOUR
T1 - Highly sensitive and multiplexed in situ rna profiling with cleavable fluorescent tyramide
AU - Xiao, Lu
AU - Labaer, Joshua
AU - Guo, Jia
N1 - Publisher Copyright:
© 2021 by the authors. Licensee MDPI, Basel, Switzerland.
PY - 2021
Y1 - 2021
N2 - 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.
AB - 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.
KW - FISH
KW - Fluorescence in situ hybridization
KW - Genomics
KW - Transcriptomics
KW - Transcripts
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U2 - 10.3390/cells10061277
DO - 10.3390/cells10061277
M3 - Article
C2 - 34063986
AN - SCOPUS:85107446810
SN - 2073-4409
VL - 10
JO - Cells
JF - Cells
IS - 6
M1 - 1277
ER -