A methodology for the combined in situ analyses of the precursor and mature forms of microRNAs and correlation with their putative targets.
A methodology for the combined in situ analyses of the precursor and mature forms of microRNAs and correlation with their putative targets.
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DOI：
10.1038/nprot.2008.215
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发表时间：
2009
期刊：
影响因子：
14.8
通讯作者：
Schmittgen, Thomas D.
中科院分区：
文献类型：
--
作者：
Nuovo, Gerard J.;Elton, Terry S.;Nana-Sinkam, Patrick;Volinia, Stefano;Croce, Carlo M.;Schmittgen, Thomas D.
目前描述的原位检测微小RNA(MiRNA)的方法相对较少，通常使用冰冻切片、信号放大和杂交或50−60°C的洗涤。MiRNA前体的检测可以通过RT原位PCR进行，从理论上讲，它可以检测到每个细胞一个拷贝。逆转录原位聚合酶链式反应的关键变量是最佳的蛋白酶消化，然后是隔夜的DNA酶消化，并将所报告的核苷酸特异性地掺入到扩增的cDNA中。成熟miRNAs的检测是通过与锁定的核酸探针进行原位杂交实现的。该方案的这一部分包括简单的蛋白酶消化，然后是隔夜杂交，短暂的低严格性洗涤和标记探针的检测。该方法的关键变量包括探针浓度和严格条件。每种miRNA原位方法需要1d。该方案的最后一步是通过免疫组织化学对miRNA的假定靶标进行标记，这是在原位杂交步骤之后进行的，需要几个小时。
There are relatively few protocols described for the in situ detection of microRNA (miRNA) and they often use cryostat sections, signal amplification and hybridization or washes of 50−60 °C. This protocol describes in situ miRNA detection that can be done in paraffin-embedded, formalin-fixed tissue. Detection of the miRNA precursors can be done by RT in situ PCR, which can theoretically detect one copy per cell. The key variable for the RT in situ PCR protocol is optimal protease digestion, which is then followed by overnight DNase digestion and target specific incorporation of the reported nucleotide into the amplified cDNA. Detection of mature miRNAs is achieved by in situ hybridization with locked nucleic acid probes. This part of the protocol involves a brief protease digestion, followed by an overnight hybridization, short low stringency wash and detection of the labeled probe. The key variables for this method include probe concentration and stringency conditions. Each miRNA in situ method takes 1 d. The final step of the protocol involves colabeling by immunohistochemistry for the putative target of the miRNA, which is done after the in situ hybridization step and takes a few hours.
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影响因子：
11.2
作者：
Gramantieri, Laura;Ferracin, Manuela;Negrini, Massimo
通讯作者：
Negrini, Massimo
影响因子：
120.7
作者：
Schetter, Aaron J.;Leung, Suet Yi;Harris, Curtis C.
通讯作者：
Harris, Curtis C.
DOI：
10.1073/pnas.0609466103
发表时间：
2006-12-12
影响因子：
11.1
作者：
Politz, Joan C. Ritland;Zhang, Fan;Pederson, Thoru
通讯作者：
Pederson, Thoru
影响因子：
11.2
作者：
Sempere, Lorenzo F.;Christensen, Mette;Cole, Charles N.
通讯作者：
Cole, Charles N.
影响因子：
14.8
作者：
Silahtaroglu, Asli N.;Nolting, Dorrit;Kauppinen, Sakari
通讯作者：
Kauppinen, Sakari