RNA FISH allows the identification and localization of noncoding RNAs in cells !
Ribonucleic Acid Fluorescence In Situ Hybridization (RNA FISH) is a visualization technique that allows simultaneous detection, localization, and quantification of individual RNA molecules at the subcellular level. Fixed or other samples can be stained or painted with fluorescently labeled probes and visualized in a widefield fluorescence microscope. A set of FISH probes may use multiple oligonucleotides with different sequences each containing a fluorescent label that collectively bind along the same target transcript to produce a localized signal.
The RNA FISH techniques is similar to the regular FISH technique in which a DNA probe is labled with a fluorescent dye whose localization will be observed under a fluorescence microscope after the hybridization reaction. The microscope provides the beam of light that excites the fluorescent tag. If a thin section of tissue is treated with a gene targeting DNA probe the probe will hybridize to the DNA in the nucleus of all the cells since they all have the same genes. The result tells the investigator that the genes are in the nucleus. However, if a probe to a virus gene is used the results will show which cells contain the virus genes, and whether the virus genes are in the cytoplasm or have penetrated the nucleus already. A chromosomal probe will allow localization of the region of a gene within a chromosome. A DNA probe can detect mRNA within the target tissue since one of the two strands of the DNA will bind the RNA. But, since the hybridization reaction needs the presence of single strands the DNA needs to be denatured by either treatment with alkali or heat. If the denaturation step is omitted only RNA will be detected.
The DNA FISH Technique
The FISH technique allows to use simple protocols, is relatively inexpensive and platform-independent, and works for many sample types and applications.
The FISH probes can be labeled with a number of various dyes. This enables multiplex detection of different RNA targets simultaneously. The nature of gene expression and RNA localization can be investigated and visualized using direct detection without isolation, purification, and amplification of the target RNA.
FISH probes for the detection of ribosomal RNA (rRNA)
Probes to detect ribosomal RNAs: The ribosomal RNAs (rRNAs) are among the most conserved macromolecules in nature and are found as an integral part of the ribosome with a high copy number in every cell. Their function is the same in every living cell on earth.
Molecular phylogenetic marker probes: The comparison of rRNA sequences allows for the reconstruction of microbial phylogeny. Many papers have been published on this subject in the past.
Probes for Bacteria and Archaea: In Bacteria and Archaea there are three types of rRNAs found in the ribosome, the 5S, 16S, and 23S rRNAs. Using comparative sequence analysis of rRNAs also allows for the identification of short signature sequences which are unique for different groups of microorganisms. These signatures can be used as targets to design specific probes with typical lengths of approximately 15 to 30 nucleotides. This type of FISH is also called “phylogenetic staining”, because it allows the identification and localization of micro-organisms in their environmental context.
FISH probes for mRNA
In the past, the simultaneous detection of multiple mRNA species in thick tissues or wholly-mounted embryos has remained technically challenging. Choi et al. in 2010 published a multiplexed fluorescent in situ hybridization method based on the triggered polymerization of RNA stem-loop structures that allows the distribution of up to five mRNAs in intact zebrafish embryos that can be imaged at the same time.
The method is based on the orthogonal amplification with hybridization chain reactions (HCR). This approach allows the use of RNA probes that are complementary to mRNA targets. The probes trigger chain reactions in which fluorescently labeled RNA hairpins self-assemble into tethered fluorescent amplification polymers. The researchers report that the HCR amplifiers exhibit deep sample penetration, high signal-to-background ratios and sharp signal localization. RNA probes, 81 nucleotides long, can be synthesized by in vitro transcription. HVR RNA hairpins, 52 nucleotides long, can be designed by considering sets of target secondary structures and synthesized chemically using the standard phosphoramidite chemistry.
FISH probes for noncoding RNA (ncRNA)
Noncoding RNAs consist of a set of RNA species with diverse roles in eukaryotic cells. Both, smaller RNA species as well as larger noncoding RNA transcripts are now thought to be abundant in mammalian cells. Hutchinson et al. in 2007 showed that RNA FISH can be used to localize RNA species in the nuclear speckles in both human and mouse cells. Their studies showed that noncoding RNAs are enriched in the nucleus and that one of these transcripts, NEAT1, localizes to the periphery of the SC35 domain of the nuclear speckle, and a neighboring transcript, NEAT2, is part of the polyadenylated component of nuclear speckles.The research group used the FISH probes to identify, localize and visualize the noncoding RNA transcripts XIST, NEAT1 and NAET2, and NEAT2/MALAT-1.
A short glossary of terms
MALAT-1 (metastasis associated lung adenocarcinoma transcript 1) also known as NEAT2 (noncoding nuclear-enriched abundant transcript 2) is a large, infrequently spliced non-coding RNA. It is highly conserved in mammals and is expressed in the nucleus. It is thought to regulate the expression of metastasis-associated genes.
NEAT stands for “nuclear enriched abundant transcript”. The Nuclear Enriched Abundant Transcript 1 (NEAT1) is a ~3.2 kb nuclear long non-coding RNA (RIKEN cDNA 2310043N10Rik) and is also known as Virus Inducible NonCoding RNA (VINC) or MEN epsilon RNA.
RNA-binding proteins (RBPs) are proteins that bind to RNA recognition motifs (RRMs) of double or single stranded RNA in cells. They are cytoplamic and nuclear proteins that participate in the formation of ribonucleoprotein complexes.
SC35 domains are found in the eukaryotic nucleus. In the nucleus the distribution of pre-mRNA splicing factors is not uniform but is reported to be concentrated at 20-40 sites. These sites with lower levels of factors diffuse throughout the nucleoplasm and are referred to as “speckles”, “SC35 domains” or “splicing factor compartments (SFCs)”. These irregular but discrete domains can be visualized with antibodies against the spliceosome assembly factor SC35. Each domain, 0.5-3.0 microns in diameter, appears to correspond largely if not entirely to ultrastructures termed interchromatin granule clusters (IGCs).
Xist (X-inactive specific transcript) is an RNA gene found on the X chromosome of placental mammals responsible for X inactivation.
Harry M T Choi, Joann Y Chang, Le A Trinh, Jennifer E Padilla, Scott E Fraser & Niles A Pierce; Programmable in situ amplification for multiplexed imaging of mRNA expression. Nature Biotechnology 28,1208–1212 (2010). doi:10.1038/nbt.1692.
Hutchinson JN, Ensminger AW, Clemson CM, Lynch CR, Lawrence JB, Chess A.; A screen for nuclear transcripts identifies two linked noncoding RNAs associated with SC35 splicing domains. BMC Genomics. 2007 Feb 1;8:39.
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