Sequential RNA and DNA fluorescence in situ hybridization (Prot 39)
An increasing body of evidence indicates that the spatial positioning of genes in the interphase nucleus is highly relevant for their function (Lanctot et al, 2007; Meaburn & Misteli, 2007; Misteli, 2007). Fluorescence in situ hybridization (FISH) is a powerful technique to map gene loci in the interphase nucleus. Depending on protocol FISH can either detect DNA or RNA. Both methods have limitations. DNA FISH only detects the physical location of a gene, but can not detect gene activity. RNA FISH, on the other hand, detects transcripts, but might miss a significant number of alleles, since not all alleles of a gene are necessarily transcribed simultaneously. The most efficient way to map gene loci and their activity is sequential RNA and DNA FISH. This is an important technique to uncover how gene positioning is linked to activity.
Simultaneous detection of RNA and DNA for a gene locus is non-trivial. Procedures during DNA FISH particularly denaturation of cellular DNA, can cause significant loss of RNA FISH signals. To overcome this problem, we have employed tyramide signal amplification (TSA) to detect RNA FISH signals in a combined DNA/RNA FISH protocol in which RNA is first detected followed by DNA detection. Since tyramide reacts with adjacent tyrosine residues in the presence of peroxidase activity and covalently binds to the residues, the RNA FISH signal is protected in the subsequent DNA FISH procedure. This protocol uses biotin-labeled single stranded DNA probes for RNA detection designed against nascent transcripts or mRNA of the gene of interest, and hybridized probes are visualized using TSA. After RNase treatment, DNA FISH is carried out with a conventional method. The signal enhancement by TSA may give rise to some background both in the nucleoplasm and remaining cytoplasm, but DNA FISH differentiates them from active loci.
With this method, we have successfully mapped active and inactive alleles of IL-4 in lymphocytes. This enabled us to compare the radial distribution of active IL-4 alleles to the inactive ones, where we found a more internal positioning of the active alleles in the interphase nucleus (Takizawa et al, 2008).
Cell Biology of Genomes, National Cancer Institute, NIH – 41 Library Drive, Bldg. 41 – Bethesda, MD 20892, USA