Riboprobe

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A Riboprobe, abbreviation of RNA probe, is a segment of labelled RNA that can be used to detect a target mRNA or DNA during in situ hybridization. [1] RNA probes can be produced by in vitro transcription of cloned DNA inserted in a suitable plasmid downstream of a viral promoter. Some bacterial viruses code for their own RNA polymerases, which are highly specific for the viral promoters. Using these enzymes, labeled NTPs, and inserts inserted in both forward and reverse orientations, both sense and antisense riboprobes can be generated from a cloned gene.

Contents

Since James Watson and Francis Crick revealed the double helix nature of DNA molecule (Watson & Crick, 1953 [2] ), the hydrogen bonds between the four bases are well known: adenine always binds to thymine and cytosine always binds to guanine. This binding pattern is the basic principle of modern genetic technologies. Joseph Gall and Mary Lou Pardue published a paper in 1969 demonstrating that radioactive marked ribosomal DNA can be used to detect its complementary DNA sequence in a frog egg, [3] known as the first researchers who use DNA probes to perform in situ hybridization. RNA probes were proved to be able to perform the same function and also used with in situ hybridization. Fluorescent dyed probes replaced radio labeled probes due to the consideration of safety, stability, and ease of detection. [4] Detecting a DNA sequence is similar to "looking for a needle in a haystack, with the needle being the DNA sequence of interest and the haystack being a set of chromosomes [5] ". The ability of the DNA helix to disassociate, re-anneal and the remarkable accuracy of base-pairing grants riboprobes the ability to locate its complementary DNA sequence on chromosomes.

It is found by two independent riboprobes, that the most important cell populations of the hippocampus, are expressing high levels of diacylglycerol lipase α (DGL-α), an enzyme involved in the production of the endocannabinoid 2-arachidonoyl-glycerol (2-AG: C 23 H 38 O 4; 20:4, ω-6). [6]

Applications

There are two kinds of probes used during in situ hybridization: Riboprobes and DNA oligonucleotides probes. [7] Riboprobes are essential in the study of embryo development in which DNA probes are insufficient. With labelled (fluoresce-dyed, for instance) antisense RNA probes hybridized with developing embryo's mRNA, tracking the expression of genes in different stages of development is possible. RNA probes can be used in detecting either whole embryo's development or just on tissue sections of interest. The ability of riboprobes to bind to transcribed mRNA makes RNA probes important in research on model organisms: Drosophila, zebrafish, chick, Xenopus and mouse. [8] RNA probes can also be utilized in immunohistochemistry to identify tissue infection in embryos. [9] Viral mRNA can be targeted by its antisense RNA probes, while infected tissues don't have complementary mRNA that can hybridize with probes; the unique mRNA sequence of each organism makes the detection of expression of certain gene highly effective and accurate.

Fluorescence in situ hybridization (FISH)is the most widely used riboprobe technique. A target sequence and a probe are essential in FISH. First, the probe is labeled with either direct or indirect labeling strategy: hapten-modified nucleotides are used in indirect labeling, and fluorophore-modified nucleotides are used in direct labeling. The target DNA and probes are denatured and mixed, allows the re-annealing of DNA sequences. Indirect labeling requires an extra step to produce visualized signals that require use of enzymatic or immunological system, but provides greater signal amplification than direct labeling. [10]

FISH probes can also be used in karyotype studies. DNA probes can be labeled with various fluorochromes which produce a unique color for each chromosomes. The probes are then hybridized with metaphase chromosomes, producing unique patterns on each chromosomes. This method is useful when people want to study the translocation, deletion and duplication of chromosomes on a larger scale comparing to site-specific FISH. [11]

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The northern blot, or RNA blot, is a technique used in molecular biology research to study gene expression by detection of RNA in a sample.

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A DNA microarray is a collection of microscopic DNA spots attached to a solid surface. Scientists use DNA microarrays to measure the expression levels of large numbers of genes simultaneously or to genotype multiple regions of a genome. Each DNA spot contains picomoles of a specific DNA sequence, known as probes. These can be a short section of a gene or other DNA element that are used to hybridize a cDNA or cRNA sample under high-stringency conditions. Probe-target hybridization is usually detected and quantified by detection of fluorophore-, silver-, or chemiluminescence-labeled targets to determine relative abundance of nucleic acid sequences in the target. The original nucleic acid arrays were macro arrays approximately 9 cm × 12 cm and the first computerized image based analysis was published in 1981. It was invented by Patrick O. Brown. An example of its application is in SNPs arrays for polymorphisms in cardiovascular diseases, cancer, pathogens and GWAS analysis. It is also used for the identification of structural variations and the measurement of gene expression.

<span class="mw-page-title-main">Fluorescent tag</span>

In molecular biology and biotechnology, a fluorescent tag, also known as a fluorescent label or fluorescent probe, is a molecule that is attached chemically to aid in the detection of a biomolecule such as a protein, antibody, or amino acid. Generally, fluorescent tagging, or labeling, uses a reactive derivative of a fluorescent molecule known as a fluorophore. The fluorophore selectively binds to a specific region or functional group on the target molecule and can be attached chemically or biologically. Various labeling techniques such as enzymatic labeling, protein labeling, and genetic labeling are widely utilized. Ethidium bromide, fluorescein and green fluorescent protein are common tags. The most commonly labelled molecules are antibodies, proteins, amino acids and peptides which are then used as specific probes for detection of a particular target.

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Fluorescence <i>in situ</i> hybridization Genetic testing technique

Fluorescence in situ hybridization (FISH) is a molecular cytogenetic technique that uses fluorescent probes that bind to only particular parts of a nucleic acid sequence with a high degree of sequence complementarity. It was developed by biomedical researchers in the early 1980s to detect and localize the presence or absence of specific DNA sequences on chromosomes. Fluorescence microscopy can be used to find out where the fluorescent probe is bound to the chromosomes. FISH is often used for finding specific features in DNA for use in genetic counseling, medicine, and species identification. FISH can also be used to detect and localize specific RNA targets in cells, circulating tumor cells, and tissue samples. In this context, it can help define the spatial-temporal patterns of gene expression within cells and tissues.

<i>In situ</i> hybridization Laboratory technique to localize nucleic acids

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<span class="mw-page-title-main">Nucleic acid test</span> Group of techniques to detect a particular nucleic acid sequence

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Flow-FISH is a cytogenetic technique to quantify the copy number of RNA or specific repetitive elements in genomic DNA of whole cell populations via the combination of flow cytometry with cytogenetic fluorescent in situ hybridization staining protocols.

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In molecular biology, hybridization is a phenomenon in which single-stranded deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) molecules anneal to complementary DNA or RNA. Though a double-stranded DNA sequence is generally stable under physiological conditions, changing these conditions in the laboratory will cause the molecules to separate into single strands. These strands are complementary to each other but may also be complementary to other sequences present in their surroundings. Lowering the surrounding temperature allows the single-stranded molecules to anneal or “hybridize” to each other.

A hybridization assay comprises any form of quantifiable hybridization i.e. the quantitative annealing of two complementary strands of nucleic acids, known as nucleic acid hybridization.

Spatial transcriptomics is a method for assigning cell types to their locations in the histological sections. It comprises an important part of spatial biology. Recent work demonstrated that the subcellular localization of mRNA molecules, for example, in the nucleus can also be studied.

References

  1. ↑ Lackie, John (2010). A Dictionary of Biomedicine. Oxford University Press. ISBN   9780199549351.
  2. ↑ Watson, J.D.; F.H.C., Crick (1953). "Molecular structure of nucleic acids: A structure for deoxyribose nucleic acid". Nature. 171 (4356): 737–738. doi:10.1038/171737a0. PMID   13054692. S2CID   4253007.
  3. ↑ Gall, J.G.; Pardue, M.L. (1969). "Formation and detection of RNA-DNA hybrid molecules in cytological preparations". Proceedings of the National Academy of Sciences. 63 (2): 378–383. doi: 10.1073/pnas.63.2.378 . PMC   223575 . PMID   4895535.
  4. ↑ Rudkin, G.T.; Stollar, B.D. (1977). "High resolution of DNA-RNA hybrids in situ by indirect immunofluorescence". Nature. 265 (5593): 472–474. doi:10.1038/265472a0. PMID   401954. S2CID   4165112.
  5. ↑ O'Connor, Clare (2008). "Fluorescence In Situ Hybridization (FISH)". Nature Education. 1: 171.
  6. ↑ Katona, István; Urbán, Gabriella M.; Wallace, Matthew; Ledent, Catherine; Jung, Kwang-Mook; Piomelli, Daniele; Mackie, Ken; Freund, Tamás F. (2006-05-24). "Molecular Composition of the Endocannabinoid System at Glutamatergic Synapses". Journal of Neuroscience. 26 (21): 5628–5637. doi:10.1523/JNEUROSCI.0309-06.2006. ISSN   0270-6474. PMID   16723519.
  7. ↑ Lajtha, Abel (2007). Handbook of Neurochemistry and Molecular Neurobiology Practical Neurochemistry Method. United States: Springer ScienceþBusiness Media. p. 364. ISBN   9780387303598.
  8. ↑ Clark, Melody (1996). In Situ Hybridization. ISBN   9783527308859.
  9. ↑ D.R., Kapczy (2001). "Detection of In Ovo-Inoculated Infectious Bronchitis Virus by Immunohistochemistry and In Situ Hybridization with a Riboprobe in Epithelial Cells of the Lung and Cloaca". Avian Diseases. 46 (3 (Jul.– Sep., 2002)): 679–685. doi:10.1637/0005-2086(2002)046[0679:doioii]2.0.co;2. S2CID   12144776.
  10. ↑ Speicher, M.R.; et al. (2005). "The new cytogenetics: blurring the boundaries with molecular biology". Nature Reviews Genetics. 6 (10): 782–92. doi:10.1038/nrg1692. PMID   16145555. S2CID   15023775.
  11. ↑ McNeil, N.; Ried, T. (2000). "Novel molecular cytogenetic techniques for identifying complex chromosomal rearrangements: technology and applications in molecular medicine". Expert Reviews in Molecular Medicine. 2000: 1–14. doi:10.1017/S1462399400001940. PMID   14585138. S2CID   196593251.

Baynes, John W.; Marek H. Dominiczak (2005). Medical Biochemistry 2nd. Edition . Elsevier Mosby. p.  477. ISBN   978-0-7234-3341-5.

YouTube video:in situ hybridization

A detailed description of Fluorescence In Situ Hybridization:

Riboprobe In Vitro Transcription Systems Technical Manual:

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