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.
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Change history
25 March 2010
In the version of this article initially published, the following text was omitted from the acknowledgments section: “We also thank Nicola Valeri, Pierliugi Gasparini and Muller Fabbri for their excellent work in providing the basis for the bcl-2/miR-16, MSH 2/miR-155 and miR-155 and Castleman’s disease associations that were an important part of the colabeling experiments. Dr Valeri is a Fellow of the American-Italian Cancer Foundation.” In addition, in Table 1, the information in the fifth row (for ref. 11) is incorrect, as described below: • In the “Probe label” column, “FITC” should be “FITC or Cy3”. • In the “Probe concentration” column, “Not listed” should be “2.5 pmol per 20 μl”. • In the “Wash” column, “37 °C, not listed” should be “37 °C, 1× SSC”. • In the “Tissue” column, “FFPE” should be “PFFCC”. • In the “Colabel/RT in situ PCR” column, “”No/No” should be “Yes/No”. Accordingly, the definition for PFFCC (permeabilized, formalin-fixed cells on coverslips) has been added to the table footnote. These errors have been corrected in the HTML and PDF versions of the article.
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Acknowledgements
We greatly appreciate the assistance and reagents from Ventana Medical Systems, and the direct assistance of Christopher Roberts, PhD, Kathleen Sergott and Margaret Nuovo, MD, who assisted with the photography and the figures. This work was supported by a grant from the Lewis Foundation (G.J.N.) and R21 CA114304 (T.D.S.).
We also thank Nicola Valeri, Pierliugi Gasparini and Muller Fabbri for their excellent work in providing the basis for the bcl-2/miR-16, MSH 2/miR-155 and miR-155 and Castleman’s disease associations that were an important part of the colabeling experiments. Dr Valeri is a Fellow of the American-Italian Cancer Foundation.
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Nuovo, G., Elton, T., Nana-Sinkam, P. et al. A methodology for the combined in situ analyses of the precursor and mature forms of microRNAs and correlation with their putative targets. Nat Protoc 4, 107–115 (2009). https://doi.org/10.1038/nprot.2008.215
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DOI: https://doi.org/10.1038/nprot.2008.215
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