Nuovo Gerard J, Elton Terry S, Nana-Sinkam Patrick, Volinia Stefano, Croce Carlo M, Schmittgen Thomas D
Department of Pathology, Ohio State University Medical Center, Columbus, Ohio, USA.
Nat Protoc. 2009;4(1):107-15. doi: 10.1038/nprot.2008.215. Epub 2009 Jan 8.
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 degrees 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.
目前针对微小RNA（miRNA）原位检测所描述的方案相对较少，并且这些方案通常使用冰冻切片、信号放大以及50 - 60摄氏度的杂交或洗涤步骤。本方案描述了一种可在石蜡包埋、福尔马林固定组织中进行的miRNA原位检测方法。miRNA前体的检测可通过逆转录原位聚合酶链反应（RT in situ PCR）来完成，理论上该方法能够检测到每个细胞中的一个拷贝。RT in situ PCR方案的关键变量是最佳蛋白酶消化，随后进行过夜脱氧核糖核酸酶（DNase）消化，并将报告核苷酸特异性掺入扩增的互补脱氧核糖核酸（cDNA）中。成熟miRNA的检测是通过与锁核酸探针进行原位杂交来实现的。该方案的这一部分包括短暂的蛋白酶消化，随后进行过夜杂交、短时间的低严谨度洗涤以及标记探针的检测。此方法的关键变量包括探针浓度和严谨度条件。每种miRNA原位检测方法需要1天时间。该方案的最后一步涉及在原位杂交步骤之后，通过免疫组织化学对miRNA的假定靶标进行共标记，这需要几个小时。
