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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Abstract
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.