Journal
NEW BIOTECHNOLOGY
Volume 61, Issue -, Pages 116-123Publisher
ELSEVIER
DOI: 10.1016/j.nbt.2020.12.001
Keywords
Fluorescence in situ hybridization; Rolling circle amplification; Padlock probes; DNA ligation; Gene expression
Funding
- National Science Foundation of China [31770927]
- National Key Research and Development Program of China [2017YFA0106800]
- Natural Science Foundation of Fujian Province [2019J01072]
- Program for Minjiang Scholar of Fujian Province
- Scientific Research Funds of Huaqiao University
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The asmFISH assay introduces a more efficient and specific method for highly specific imaging of individual transcripts in fixed cells and tissues, compared to the padlock probe assay. It can be applied not only to cultured cells, but also to fresh frozen and formalin-fixed, paraffin-embedded tissue sections.
An amplification-based single-molecule fluorescence in situ hybridization (asmFISH) assay is introduced that exploits improved probe design for highly specific imaging of individual transcripts in fixed cells and tissues. In this method, a pair of DNA ligation probes are ligated on RNA templates upon specific hybridization, followed by probe circularization based on enzymatic DNA ligation and rolling circle amplification for signal boosting. The method is more efficient and specific than the padlock probe assay for detection of the same RNA molecules and discrimination of single nucleotide polymorphisms. Moreover, asmFISH is a versatile method which can be applied not only to cultured cells, but also to fresh frozen and formalin-fixed, paraffin-embedded tissue sections.
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