In Situ Hybridization Radiolabeled

Question: Discuss about theIn Situ Hybridization forRadiolabeled. Answer: Introduction: In Situ hybridization can be defined as a technique which involves labeling of a probe consisting of one or more than one genes with radiolabeled or fluorescent dye or a reporter molecule is hybridized to specific cells resulting in a coloured product. This technique uses a labeled complementary DNA, RNA or any other nucleic acid strand in order to localize a particular DNA or RNA sequence (1). The probes generally used in this technique are dsDNA probe, ssDNA probe, RNA probe and synthetic oligonucleotides probe. This report aims to focus on the application, advantages or disadvantages and control measures involved in the technique (2). Application: In Situ hybridization have high significance in the application of locating specific nucleic acid sequences on tissues or chromosomes in order to understand the regulation, organization and specific gene function (5). The various applications of In Situ Hybridization are as follows: To identify the population structure and morphology of microorganisms. This technique is also used in the pathological study such as abnormal gene expression and pathogen profiling. In Situ Hybridization is also applied in phylogenetic analysis and karyotyping. It is also involved in the study of developmental biology such as gene expression profiling. This technique is used in physical mapping (5). Figure: Generation of Riboprobes using In Situ Hybridization (Source: www.ncbi.nlm.nih.gov) Advantages or Limitations: The main advantage of In Situ Hybridization is that, the technique aids to determine the distribution of specific nucleic acids in relation to the protein products of the gene target. It also enables to identify the relation of the nucleic acids with cellular structures with the application of immunochemistry (4). Moreover, EFTEM technique produces images that can give quantitative information. However, the major disadvantage of In Situ Hybridization is the difficulty in the process of determining targets with low DNSA or RNA copies. Moreover, its time consuming and costly. Data Collected From the Approach: The data generated by FISH depends on the sources of variability during the analysis process (3). The sources of variation might arise from the differences in probe used, sample preparation, hybridization protocols, heterogeneity of intrasample, filters and counting criteria. The fluorescently labeled probe that has hybridized to the nucleus in the cell of interest will appear as specific fluorescent dot. This implicates the success of the hybridization technique using Fluorescent In Situ Hybridization (FISH). Analytical validation allows the interpretation of the outcome of the FISH data. In addition to this the high degree of probe specificity aids in better interpretation of data in terms of FISH (2). Controls Used: In terms of In Situ Hybridization controls although controls are inclusion of such experiments, they are not used properly. Controls for tissue mRNA quality are added to verify the status of mRNA and tissue (5). In Situ Hybridization controls are of two types, positive and negative controls. Polyd (T) is generally included in the control. Sometimes in certain experiments in situ hybridization is performed using the proper oligonucleotide probe on a new, positive control tissue which is known to contain the sequence of interest (1). Conclusion: In Situ Hybridization is a powerful tool used for localizing of a particular nucleic acid targets within a cell or tissue that helps in the obtaining information about the gene expression and gene loci. The fluorescently labeled probe that has hybridized to the nucleus in the cell of interest will appear as specific fluorescent dot and this data re interpreted to validate the required outcome. There are various applications of In Situ Hybridization such as in the field of microbiology, immunology and pharmacogenomics. However, the limitation of this technique is it is costly and time consuming. References: Murray PR, Rosenthal KS, Pfaller MA. Medical microbiology. Elsevier Health Sciences; 2015 Oct 28. Nagarajan K, Loh KC. Molecular biology-based methods for quantification of bacteria in mixed culture: perspectives and limitations. Applied microbiology and biotechnology. 2014 Aug 1;98(16):6907-19. Nikolakakis K, Lehnert E, McFall-Ngai MJ, Ruby EG. Use of hybridization chain reaction-fluorescent in situ hybridization to track gene expression by both partners during initiation of symbiosis. Applied and environmental microbiology. 2015 Jul 15;81(14):4728-35. Stender H, Williams B, Coull J. PNA fluorescent in situ hybridization (FISH) for rapid microbiology and cytogenetic analysis. Peptide Nucleic Acids: Methods and Protocols Second Edition. 2014:167-78. Wagner M, Haider S. New trends in fluorescence in situ hybridization for identification and functional analyses of microbes. Current opinion in biotechnology. 2012 Feb 29;23(1):96-102. Wright ES, Yilmaz LS, Corcoran AM, kten HE, Noguera DR. Automated design of probes for rRNA-targeted fluorescence in situ hybridization reveals the advantages of using dual probes for accurate identification. Applied and environmental microbiology. 2014 Aug 15;80(16):5124-33.