In Situ Hybridization


Presentation Description

In situ hybridization is a powerful technique for identifying specific mRNA species within individual cells in tissue sections, providing insights into physiological processes and disease pathogenesis. However, in situ hybridization requires that many steps be taken with precise optimization for each tissue examined and for each probe used.


Presentation Transcript

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In Situ Hybridization

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CONTENT NO.1 ISH NO.2 CISH NO.3 FISH NO.4 General Procedure

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In Situ Hybridization In Situ Hybridization ISH is a technique that allows for precise localization of a specific segment of nucleic acid within a histologic section. The underlying basis of ISH is that nucleic acids if preserved adequately within a histologic specimen can be detected through the application of a complementary strand of nucleic acid to which a reporter molecule is attached. Introduction In Situ Hybridization

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Probes Double-stranded DNA dsDNA probes Single-stranded DNA ssDNA probes RNA probes riboprobes Synthetic oligonucleotides PNA LNA Probes - ISH

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Probe types and their pros and cons Probe Types Advantages Disadvantages Double-stranded DNA dsDNA probes Stable available easier to obtain Self-hybridize less sensitive need denaturation before hybridization Single-stranded DNA ssDNA probes Stable easier to work with more specific resistant to RNases better tissue penetration without self-hybridize Time consuming expensive RNA probes riboprobes Higher thermal stability better tissue penetration more specific low background noise by RNase Sensitive to RNases Synthetic oligonucleotides Economical stable available easier to work with more specific resistant to RNases better tissue penetration better reproducibility Know the information of nucleotide sequence Probes - ISH

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Labeling Techniques - ISH Probe Labeling and Signal Detection ✓ 32 P ✓ 35 S ✓ 3 H ✓ Biotin ✓ Digoxigenin ✓ Fluorescent dye FISH Radioactive isotopes Non-radioactive labels

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Chromogenic In Situ Hybridization CISH CISH enables examination of gene amplification gene deletion chromosomal translocations and chromosomal number. This approach uses conventional peroxidase or alkaline phosphatase reactions using bright-field microscopy on tissues fixed by formalin and embedded in paraffin. These peroxidase or alkaline phosphatase-labeled reporter antibodies interact with a hybridized DNA probe and are then observed with an enzymatic reaction. Tissue morphology and genetic abnormalities can be viewed at the same time with CISH. Introduction of CISH

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Variations - CISH SISH uses a similar method as CISH but a silver precipitate is the end product rather than a brown product. Silver-enhanced in situ hybridization SISH

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1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 DuoCISH is a variation of CISH that addresses the need for two different probes on the same slide. It is a well- established technique for HER- 2/neu amplification detection even though it is sometimes reported to be less effective than FISH. DuoCISH Variations - CISH

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Medical Applications Gene amplification CISH is frequently applied to assess gene amplification such as HER-2/neu status in breast cancer samples. Chromosomal rearrangements CISH is also used for detection of chromosomal rearrangements and fusions such as the fusion of ALK tyrosine kinase domain with the promoter and 5’ region of EML4 in lung cancer. Papillomavirus infections Apart from cancers CISH has also been shown to be useful in detecting human papillomavirus infections. Chromogenic In Situ Hybridization CISH

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Fluorescence In Situ Hybridization FISH Fluorescence in situ hybridization FISH is a molecular cytogenetic technique that uses fluorescent probes that bind to only those parts of a nucleic acid sequence with a high degree of sequence complementarity.

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Single-molecule RNA FISH is a method of detecting and quantifying mRNA and other long RNA molecules in a thin layer of tissue sample. Single-molecule RNA FISH assays can be performed in simplex or multiplex and can be used as a follow- up experiment to quantitative PCR or imaged simultaneously with a fluorescent antibody assay. The technology has potential applications in cancer diagnosis neuroscience gene expression analysis and companion diagnostics. Single-molecule RNA FISH Variations - FISH

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Variations - FISH In an alternative technique to interphase or metaphase preparations fiber FISH interphase chromosomes are attached to a slide in such a way that they are stretched out in a straight line rather than being tightly coiled as in conventional FISH or adopting a chromosome territory conformation as in interphase FISH. This is accomplished by applying mechanical shear along the length of the slide either to cells that have been fixed to the slide and then lysed or to a solution of purified DNA. Fiber FISH

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Q-FISH MA-FISH Variations - FISH Microfluidics-assisted FISH MA-FISH uses a microfluidic flow to increase DNA hybridization efficiency decreasing expensive FISH probe consumption and reduce the hybridization time. MA-FISH is applied for detecting the HER2 gene in breast cancer tissues. Q-FISH combines FISH with PNAs and computer software to quantify fluorescence intensity. This technique is used routinely in telomere length research.

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Hybrid Fusion FISH HF-FISH uses primary additive excitation/emission combination of fluorophores to generate additional spectra through a labeling process known as dynamic optical transmission DOT. Three primary fluorophores are able to generate a total of 7 readily detectable emission spectra as a result of combinatorial labeling using DOT. Hybrid Fusion FISH enables highly multiplexed FISH applications that are targeted within clinical oncology panels. Hybrid Fusion-FISH Variations - FISH

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Species Identification Comparative Genomic Hybridization Virtual Karyotype Spectral Karyotype Medical Applications -FISH

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General Procedure - Select appropriate BAC for target of interest extract and amplify DNA - Label probe with biotin or digoxigenin using random priming or nick translation Probe Design - Fix tissue to a glass slide using paraffin - Wash and heat slide several times to remove paraffin from surface - Pepsin digestion to ensure access to target DNA sequence Tissue Prep - Add 10-20 uL probe to tissue cover slide and seal coverslip - Heat slide to 97℃ for 5-10 min to denature DNA - Place slide in 37℃ oven overnight for probe to hybridize Hybridization - Add a blocker to bind nonspecific binding sites - Add hydrogen peroxide to suppress endogenous peroxidase activity Blocking - - If digoxigenin is the label: add anti-digoxigenin fluorescein primary antibody followed by a HRP-conjugated anti-fluorescein secondary antibody - - If biotin is the label: HRP-conjugated streptavidin is used for detection - - Add DAB which is converted to a brown precipitate by HRP Probe Detection

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