Simultaneous RNA–DNA FISH in Mouse Preimplantation Embryos
Fluorescent in situ hybridization (FISH) is a powerful cytogenetic technique that allows the visualization and quantification of RNA and DNA molecules in different cellular contexts. In general, FISH applications help to advance research, cytogenetics, and diagnostics. DNA FISH can be applied, for example, for gene mapping and for detecting genetic aberrations. RNA FISH provides information about gene expression. However, in cases where RNA and DNA molecules need to be detected in the same sample, the result is often compromised by the fact that the tissue sample is damaged due to the multitude of processing steps that are required for each application. In addition, the sequential application of RNA and DNA FISH protocols on the same sample is very time consuming. Here we describe a brief protocol that enables the combined and simultaneous detection of Xist RNA and centromeric DNA of chromosome 6 in mouse preimplantation embryos. In addition, we describe how to generate indirect-labeled probes starting from BACs. This protocol may be applied to any combination of RNA and DNA detection.
Key wordsRNA and DNA FISH Combination of RNA and DNA FISH Fluorescent in situ hybridization (FISH) Mouse preimplantation embryos Xist FISH X chromosome inactivation Long noncoding RNA FISH DNA FISH Long noncoding RNAs
- 13.Chaumeil J, Augui S, Chow JC, Heard E (2008) Combined immunofluorescence, RNA fluorescent in situ hybridization, and DNA fluorescent in situ hybridization to study chromatin changes, transcriptional activity, nuclear organization, and X-chromosome inactivation. Methods Mol Biol 463:297–308. https://doi.org/10.1007/978-1-59745-406-3_18CrossRefPubMedPubMedCentralGoogle Scholar
- 16.Sommerlad C, Mehraein Y, Giersberg M, Marben K, Rieder H, Rehder H (2002) Formalin-fixed and paraffin-embedded tissue sections. In: Rautenstrauss B, Liehr TH (eds) FISH technology (springer lab manuals). Springer, Berlin, Heidelberg, New York, pp 149–161Google Scholar
- 18.Green MR, Sambrook J (2012) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory press, Cold Spring HarborGoogle Scholar
- 19.Veuskens J, Hinnisdaels A, Mouras A (1993) In situ hybridization to plant metaphase chromosomes: radioactive and non-radioactive detection of repetitive and low copy number genes. In: Lindsey K (ed) Plant tissue culture manual, vol D8. Springer, New York, pp 1–15Google Scholar