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Rapid in situ codetection of noncoding RNAs and proteins in cells and formalin-fixed paraffin-embedded tissue sections without protease treatment


Noncoding RNAs (ncRNAs) comprise a diverse group of RNAs that function in essential cellular processes such as pre-mRNA splicing and mRNA translation and also regulate various aspects of gene expression in physiology and development. Methods of subcellular and tissue localization of ncRNAs are essential to understand their biological roles and their contribution to disease. We describe a rapid fluorescent (FISH) or chromogenic (CISH) in situ hybridization protocol for localization of ncRNAs (including microRNAs (miRNAs), small nucleolar RNAs (snoRNAs), small nuclear RNAs (snRNAs), piwi-associated RNAs (piRNAs) and ribosomal RNAs (rRNAs)) in formalin-fixed, paraffin-embedded (FFPE) tissues and cultured cells, using locked nucleic acid (LNA)-modified oligonucleotides. In this protocol, sections are heated in citrate buffer, which eliminates the need for protease treatment, thus preserving optimal morphology and protein epitopes, and allowing the simultaneous detection of proteins with immunofluorescence staining (IF). LNA–FISH requires 5 h, or between 10 and 36 h when combined with IF; LNA–CISH requires 2 d.

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Figure 1
Figure 2: Codetection of 18S rRNA and Sm proteins in HeLa cells and FFPE mouse tissues using directly labeled LNA probes for FISH, combined with IF.
Figure 3: Codetection of miR-124 and neuron-specific class III b-tubulin (TuJ-1) in FFPE mouse brain by LNA–FISH with TSA amplification and IF, respectively.
Figure 4: Representative FISH micrographs of different ncRNAs in mouse FFPE tissues using directly labeled LNA probes.
Figure 5: LNA–CISH for piRNA (XL-piR-3) in Xenopus laevis oocyte.

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We are grateful to the members of our laboratory and to Francesco Lotti for stimulating discussions and to Gideon Dreyfuss for providing the Y12 antibody. Supported by NIH grants GM0720777 and NS056070 to Z.M.

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M.d.P.-S. developed the protocols and performed all the experiments. M.d.P.-S., M.C.R. and Z.M. contributed to the design and analysis of experiments and wrote the paper.

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Correspondence to Zissimos Mourelatos.

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The authors declare no competing financial interests.

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de Planell-Saguer, M., Rodicio, M. & Mourelatos, Z. Rapid in situ codetection of noncoding RNAs and proteins in cells and formalin-fixed paraffin-embedded tissue sections without protease treatment. Nat Protoc 5, 1061–1073 (2010).

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