RRC ID 74823
著者 Kitazawa S, Haraguchi R, Takaoka Y, Kitazawa R.
タイトル In situ sequence-specific visualization of single methylated cytosine on tissue sections using ICON probe and rolling-circle amplification.
ジャーナル Histochem Cell Biol
Abstract Since epigenetic modifications differ from cell to cell, detecting the DNA methylation status of individual cells is requisite. Therefore, it is important to conduct "morphology-based epigenetics research", in which the sequence-specific DNA methylation status is observed while maintaining tissue architecture. Here we demonstrate a novel histochemical technique that efficiently shows the presence of a single methylated cytosine in a sequence-dependent manner by applying ICON (interstrand complexation with osmium for nucleic acids) probes. By optimizing the concentration and duration of potassium osmate treatment, ICON probes selectively hybridize to methylated cytosine on tissue sections. Since the elongation process by rolling-circle amplification through the padlock probe and synchronous amplification by the hyperbranching reaction at a constant temperature efficiently amplifies the reaction, it is possible to specifically detect the presence of a single methylated cytosine. Since the ICON probe is cross-linked to the nuclear or mitochondrial DNA of the target cell, subsequent elongation and multiplication reactions proceed like a tree growing in soil with its roots firmly planted, thus facilitating the demonstration of methylated cytosine in situ. Using this novel ICON-mediated histochemical method, detection of the methylation of DNA in the regulatory region of the RANK gene in cultured cells and of mitochondrial DNA in paraffin sections of mouse cerebellar tissue was achievable. This combined ICON and rolling-circle amplification method is the first that shows evidence of the presence of a single methylated cytosine in a sequence-specific manner in paraffin sections, and is foreseen as applicable to a wide range of epigenetic studies.
巻・号 159(3)
ページ 263-273
公開日 2023-3-1
DOI 10.1007/s00418-022-02165-2
PII 10.1007/s00418-022-02165-2
PMID 36418613
PMC PMC10006048
MeSH Animals Cytosine* DNA Methylation DNA, Mitochondrial Epigenesis, Genetic Mice Paraffin*
IF 3.418
リソース情報
ヒト・動物細胞 ST2(RCB0224) RAW 264(RCB0535)