Name: ish probes for dna and rna detection
Uploaded: 2021-04-07T12:12:00.000Z
Duration: 2 min 31 s
Channel: JoVE

K12 Standards

Tennessee Academic Standards

High School

Biology

links-among-engineering-technology-science-and-society

Links Among Engineering, Technology, Science, and Society

BIO1.ETS2.1

In-situ Hybridization

A hibridização in situ é uma técnica usada para localizar DNA ou RNA em células cultivadas ou em seções de tecido quimicamente preservadas.
A hibridização in situ de RNA é usada para monitorar onde um gene está sendo expresso, detectando e quantificando os transcritos específicos de mRNA.
Os mRNAs alvo são detectados por sondas de RNA complementares de fita simples que são criadas pela transcrição in vitro de um molde de DNA ou sintetizadas por uma reação química.
As sondas são marcadas com isótopos radioativos, fluoróforos ou outras moléculas repórteres, como biotina ou digoxigenina, que podem ser ligadas e detectadas por um anticorpo marcado.
A hibridização in situ de RNA tem quatro etapas principais: fixação do tecido, permeabilização, hibridização e detecção.
Para começar, o tecido fresco é colhido e tratado quimicamente para interromper quaisquer reações bioquímicas dentro do tecido e preservar sua integridade estrutural. Essa técnica de preservação é chamada de fixação de tecido.
Em seguida, proteínas e lipídios no tecido precisam ser removidos para que a sonda acesse e se ligue ao RNA alvo.
Para degradar as proteínas e permeabilizar a membrana celular, a amostra é tratada com ácido clorídrico 0,2 molar e enzimas como proteinases, enquanto detergentes são usados para quebrar os lipídios.
O RNA alvo é então hibridizado com as sondas a uma temperatura logo abaixo do ponto de fusão do híbrido RNA-sonda. Isso ajuda as sondas a recozer com os mRNAs complementares. As sondas não ligadas e frouxamente ligadas são removidas realizando várias lavagens.
O método para detectar as sondas hibridizadas depende do tipo de rótulo. As sondas radioativas são expostas a filmes fotográficos, enquanto as sondas fluorescentes são visíveis sob microscópios fluorescentes.
Para outros rótulos, os anticorpos contra a molécula repórter são marcados com um corante fluorescente ou colorimétrico.
A detecção de sondas hibridizadas revela a distribuição de mRNAs específicos indicando onde os genes de interesse são expressos.

<ol>
	<li>Jensen E. Revisão técnica: hibridização in situ. O registro anatômico. Maio de 2014. https://doi.org/10.1002/ar.22944</li>
	<li>Höfler, Heinz. "O que há de novo na "hibridização in situ"." Patologia-Pesquisa e Prática 182, nº 3 (1987): 421-430.</li>
</ol>

hibridização in-situ

A hibridização in situ (ISH) é uma técnica usada para detectar e localizar moléculas específicas de DNA ou RNA em células, tecidos ou seções de tecido usando uma sonda marcada. A técnica foi usada pela primeira vez em 1969 para a investigação de ácidos nucléicos. Atualmente é uma ferramenta essencial na investigação científica e em contextos clínicos, especialmente para fins de diagnóstico.

<h4>Tipos de sondas e rótulos </h4> 

Uma sonda é uma fita complementar de DNA ou RNA que se liga às sequências de nucleotídeos correspondentes em uma célula. Muitas sondas diferentes, tais como sondas de DNA de cadeia simples, sondas de DNA de cadeia dupla, sondas de ARN anti-sentido ou ribossomas e sondas de oligodesoxinucleótidos sintéticas, são utilizadas na hibridização in situ. A escolha da sonda depende de vários fatores, incluindo sensibilidade, especificidade, estabilidade e facilidade de penetração na amostra de tecido. 

Estas sondas podem ser marcadas com radioisótopos, corantes fluorescentes ou outras moléculas de antígeno para fins de detecção. O 3H, 35S e 32P são sondas radiomarcadas amplamente utilizadas, enquanto os marcadores não radioativos incluem biotina, digoxigenina e fluoresceína. Esses marcadores podem ser anexados à molécula de DNA da sonda por meio de métodos de marcação final, tradução de corte ou síntese de primer aleatório. Métodos de detecção, como autorradiografia, microscopia de fluorescência ou imuno-histoquímica, são usados para visualização do alvo com base no rótulo anexado à sonda hibridizada. <h4>Vantagens e desvantagens da hibridização in situ</h4> 

Uma das principais vantagens da hibridização in situ é que ela pode até ser aplicada em tecidos congelados para permitir o máximo aproveitamento de tecidos difíceis de obter. Além disso, pode ser combinado com outras técnicas, como a imunohistoquímica, para detectar proteínas e mRNA ativo na amostra. No entanto, ao trabalhar com amostras que possuem baixas cópias de DNA e RNA, pode ser difícil identificar alvos utilizando a hibridização in situ.

Hibridização in-situ

A hibridização in situ (ISH) é uma técnica usada para detectar e localizar moléculas específicas de DNA ou RNA em células, tecidos ou seções de tecido usando um ...

Education

JoVE Core

Molecular Biology

Biologia Molecular

Unit 2

Unidade 2

Analyzing Gene Expression and Function

Analisando a Expressão e a Função Gênica

KEY TERMS AND CONCEPTS

TERMOS-CHAVE E CONCEITOS

ish probes for dna and rna detection

In situ hybridization (ISH) uses labeled probes to detect and localize specific DNA or RNA molecules in cells, tissues, or tissue sections. The probe is a complementary strand of DNA or RNA that binds to a matching nucleotide sequence in the sample. Since its first use in 1969 for nucleic acid studies, ISH has become an important tool in research and in clinical diagnosis.

Several probe types can be used in ISH. These include single-stranded DNA probes, double-stranded DNA probes, antisense RNA probes, also called riboprobes, and synthetic oligodeoxynucleotide probes. The best choice depends on sensitivity, specificity, stability, and how easily the probe can enter the tissue sample.

Probes can also carry different labels for detection. Common labels include radioisotopes, fluorescent dyes, and other antigen molecules. Widely used radiolabeled probes include 3H, 35S, and 32P. Non-radioactive labels include biotin, digoxigenin, and fluorescein.

These labels are attached to the DNA probe by end-labeling, nick-translation, or random primer synthesis. After the probe binds to its target, the signal can be seen with autoradiography, fluorescence microscopy, or immunohistochemistry, depending on the label. ISH can even be used on frozen tissues, which helps when tissue samples are hard to obtain. It can also be combined with techniques such as immunohistochemistry to detect protein and active mRNA in the same sample. However, targets with low DNA or RNA copy number can be difficult to find with this method.

ISH Probes for DNA and RNA Detection

in vitro mutagenesis

In Vitro Mutagenesis for Gene Analysis

In vitro mutagenesis is a lab method used to change DNA and study gene function. It helps scientists test how specific DNA changes affect a gene’s activity and the traits it controls. A common approach is to make a known change in a DNA sequence and then examine the result. This can be done by deleting, inserting, or altering selected bases in a gene. The method is useful for linking a DNA change to a biological effect. Researchers use in vitro mutagenesis in molecular biology, genetics, and...

genetic screens

Using Genetic Screens to Find Disease Genes

Genetic screens are tools used to find genes and mutations linked to specific traits. They are also used to identify people or groups at risk for genetic diseases. That can support early intervention, targeted therapy, and reproductive choices. Forward genetic screens, also called classical screens, start with random DNA changes in an organism. Researchers may use radiation, mutagens, or added bases to create mutations that lead to visible phenotype changes. The mutant is then inbred so...

test cross

Test Cross in Genetic Inheritance

A test cross is a genetics tool used to determine the genotype of an organism with a dominant trait. It helps reveal whether the dominant individual is homozygous or heterozygous by crossing it with a recessive individual. The outcome of the cross shows the hidden genotype. If any offspring show the recessive trait, the dominant parent must carry one recessive allele. If all offspring show the dominant trait, the dominant parent is likely homozygous dominant. Test crosses are common in basic...

complementation tests

Using Crosses to Group Mutations

A complementation test uses a simple cross to find out whether two mutations are in the same gene or in different genes. It helps show how mutations are arranged on chromosomes. Edward Lewis first performed this test in the 1940s while studying fruit flies. To do the test, organisms heterozygous for different mutations are crossed pairwise in all combinations. If the mutations are in different genes, they can complement each other. In that case, each mutation provides the missing genetic...

single nucleotide polymorphisms-snps

SNPs as Genetic Markers and Variants

Single nucleotide polymorphisms, or SNPs, are small DNA changes at one specific genomic position. They are the most common type of sequence variation in the human genome. A point mutation counts as an SNP when it appears in more than 1% of the population.

bacterial transformation

Bacterial Transformation in the Lab

Bacterial transformation is a lab method used to move new DNA into bacteria. Scientists use it to change how bacteria behave or to help them carry a gene of interest. In high school biology, this process is often shown with plasmids, which are small circular DNA molecules. During transformation, bacteria take up DNA from their surroundings. The new DNA can then be used by the cell, depending on the experiment. This technique is an important part of genetic engineering and is widely used in...

transgenic organisms

Making Transgenic Organisms

Transgenic organisms are living things with DNA added from another organism. A transgene is the inserted gene. Scientists use this method to change an organism’s traits and to study how genes work. To make a transgenic organism, researchers first identify the gene they want to add. They then place that gene into the target organism’s DNA. Once the gene is integrated, the organism can pass it to new cells as it grows. This approach is useful in biology because it helps scientists analyze gene...

reproductive cloning

Cloning an Organism by Reproduction

Reproductive cloning creates a new organism that is genetically the same as the donor. It is different from cloning genes or cells because the goal is to make a whole living individual. This process usually relies on somatic cell nuclear transfer. In this method, the nucleus from a body cell is placed into an egg cell that has had its own nucleus removed. The egg can then develop into an embryo with DNA from the donor cell. Reproductive cloning is used to produce a copy of an organism, such...

crispr

CRISPR Gene Editing Basics

CRISPR is a gene editing tool that helps scientists make targeted changes to DNA. It uses a guide system to find a chosen sequence and a cutting protein to act at that spot. This method is based on a natural defense system found in bacteria. In high school biology, it is often studied as a way to see how genes can be edited with precision. CRISPR is widely used in molecular biology research. It helps scientists explore how genes function and how DNA changes can affect living things.

experimental rnai

RNAi for Gene Silencing and Discovery

RNA interference (RNAi) is a gene-silencing process used to reduce gene expression. It can block transcription or trigger RNA degradation. Scientists first discovered this mechanism in plants in 1998 through Andrew Fire and Craig Mello. RNAi has since been found in many eukaryotes, including protozoa, flies, nematodes, insects, parasites, and mammals. Researchers use it as a practical tool to study gene function. It can show what a gene does without permanently changing the organism’s DNA. <br...

reporter genes

Reporter Genes in Cells and Plasmids

Reporter genes are protein-coding genes used to track gene activity in cells. They are often tagged to a gene of interest so scientists can see whether that gene is present or active. When the tagged genes are expressed together, reporter genes can produce visible signals such as fluorescence or luminescence. These signals help show the presence or absence of a gene of interest in an organism. They can also reveal the pattern of gene expression or the location of a DNA segment or protein...

in-situ hybridization

In situ hybridization (ISH) uses labeled probes to detect and localize specific DNA or RNA molecules in cells, tissues, or tissue sections. The probe is a complementary strand of DNA or RNA that binds to a matching nucleotide sequence in the sample. Since its first use in 1969 for nucleic acid studies, ISH has become an important tool in research and in clinical diagnosis. Several probe types can be used in ISH. These include single-stranded DNA probes, double-stranded DNA probes, antisense...

chromatin immunoprecipitation- chip

ChIP Methods for DNA-Protein Binding

Chromatin immunoprecipitation, or ChIP, is an antibody-based method for finding where proteins bind to DNA. It is used to identify DNA sites bound by transcription factors or histone proteins. ChIP also helps show which histone changes are present, including acetylation, phosphorylation, and methylation. ChIP is commonly divided into two forms: X-ChIP and N-ChIP. X-ChIP first cross-links proteins to DNA inside the cell, then breaks the DNA into pieces by sonication. The protein-DNA complexes...

synthetic biology

Synthetic Biology in Crops and Cells

Synthetic biology combines engineering, molecular biology, cell biology, and systems biology to redesign living systems. Scientists use it to remodel organisms from nature or to build new synthetic organisms. These tools can support protein or enzyme production, bioremediation, the production of useful macromolecules, and the addition of helpful traits to crops. One well-known example is golden rice. This genetically modified rice plant makes grains rich in β-carotene, which is a precursor of...

ribosome profiling

Ribosome Profiling for Translation Snapshot

Ribosome profiling is a deep sequencing method that gives a snapshot of active translation in a cell. It sequences the mRNAs protected by ribosomes. This shows which messages are being read to make proteins at a specific moment. The method has several uses in biology. It can track translation in a living organ or tissue type. It also helps measure new protein synthesis levels. Researchers use it to find translated open reading frames, which are stretches of RNA that can make protein. It can...

transgenic plants

Transgenic Plants for Better Crops

Transgenic plants are crops that have been changed with recombinant DNA technology, also called transgenesis. This method adds a foreign gene or removes a harmful one from an organism. The result is a genetically modified organism. The first transgenic plant was a tobacco plant made in 1983, and it was resistant to tobacco mosaic virus. Since then, transgenic plants have been developed for many uses in agriculture, horticulture, and ornamental plants. Some are designed to resist pests or...

gene therapy

Gene Therapy for Disease Treatment

Gene therapy is a treatment approach that changes gene activity to help address disease. It focuses on the genetic cause of a problem rather than only easing symptoms. In this way, it is used in disease intervention. One common strategy is to replace a faulty gene with a normal copy. Another strategy is to inactivate a gene that is causing harm. Gene therapy can also introduce a new gene to help cells work more normally.

epistasis analysis

Gene Interaction Maps in Epistasis

Gene interaction maps in epistasis show how different genes work together to shape a phenotype. In epistasis, the interaction of genes or alleles at different locations affects the final trait. This often happens when one gene masks or interferes with the expression of another gene. Epistasis is common when genes are part of the same biochemical pathway. In that case, the expression of one gene can depend on a gene product made earlier in the pathway. Genes that mask or inhibit other genes in...

genome-wide association studies-gwas

Using SNP Patterns to Link Disease Risk

Genome-wide association studies, or GWAS, look for common SNPs, or single nucleotide polymorphisms, that may be linked to disease. Researchers compare how often a SNP appears in people with a disease and in people without it. When a SNP is seen more often in the disease group, it is considered associated with that disease. GWAS uses chi-square analysis to test the chance that an allele is linked to the disease. This approach does not require scientists to first find the target gene behind the...

key terms and concepts

Learn how in situ hybridization uses labeled DNA and RNA probes to find specific sequences in cells and tissues, with common labels and detection methods.

ISH Probes for DNA and RNA Detection | JoVE K12

molecular biology

JoVE Core Biologia Molecular mostra os processos moleculares da célula através de vídeos de animação claros e concisos. Além disso, nos vídeos de Cientistas em ação, a aplicação desses conceitos é demonstrada por meio de experimentos reais em laboratórios modernos.

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An Overview of Genetic Engineering

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Recombineering and Gene Targeting

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Genome Editing

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Recombinant DNA

DNA Ligation Reactions

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Restriction Enzyme Digests

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DNA Isolation

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Restriction Enzymes

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DNA Agarose Gel Electrophoresis

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DNA Gel Electrophoresis

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Separating Protein with SDS-PAGE

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Plasmid Purification

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Gel Purification

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PCR - Polymerase Chain Reaction

Real Time RT-PCR

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RACE - Rapid Amplification of cDNA Ends

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PCR: The Polymerase Chain Reaction

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bacterial-transformation-the-heat-shock-method

Bacterial Transformation: The Heat Shock Method

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Bacterial Transformation: Electroporation

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An Introduction to Transfection

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Labeling DNA Probes

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Southern Blot

Agarose gel electrophoresis is very useful in separating DNA fragments by size. Running a DNA ladder containing fragments of the known length alongside ...

DNA Microarrays

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An Overview of Gene Expression

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Expression Profiling with Microarrays

Microarrays are important tools for profiling gene expression, and are based on complementary binding between probes that are attached to glass chips and ...

Complementary DNA

FISH - Fluorescent In-situ Hybridization

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Sanger Sequencing

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Maxam-Gilbert Sequencing

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Next-generation Sequencing

The first human genome sequencing project cost $2.7 billion and was declared complete in 2003, after 15 years of international cooperation and ...

Chromatin Immunoprecipitation- ChIP

Chromatin immunoprecipitation, or ChIP, is an antibody-based technique used to identify sites on DNA that bind to transcription factors of interest or ...

RNA-seq

RNA sequencing, or RNA-Seq, is a high-throughput sequencing technology used to study the transcriptome of a cell. Transcriptomics helps to interpret the ...

Synthetic Biology

Synthetic biology is an interdisciplinary science that involves using principles from disciplines such as engineering, molecular biology, cell biology, ...

Genome Annotation and Assembly

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Ribosome Profiling

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The ELISA Method

An enzyme-linked immunosorbent assay (ELISA) is typically performed to detect the presence and/or amount of a target protein of interest within an ...

Transgenic Plants

Recombinant DNA technology called transgenesis is often used to add a foreign gene or remove a detrimental gene from an organism. Such genetically ...

Transgenic Organisms

Gene Therapy

Reproductive Cloning

CRISPR

Experimental RNAi

RNA interference (RNAi) is a cellular mechanism that inhibits gene expression by suppressing its transcription or activating the RNA degradation process. ...

Reporter Genes

Reporter genes are a type of&#160;protein-coding gene&#160;that are often tagged to a gene of interest. Once inside a target cell, reporter genes usually ...

In situ hybridization (ISH) is a technique used to detect and localize specific DNA or RNA molecules in cells, tissue, or tissue sections using a labeled ...

Obtain, evaluate, and communicate information on how molecular biotechnology may be used in a variety of fields.
EXPLANATION: The goal is to help students appreciate that the basic science knowledge being studied can be manipulated and used to design useful tools for further scientific investigations, medical treatments, agricultural yields, etc. A detailed understanding of any technical procedures is not expected, but rather a "big picture" view of concepts the technology utilizes and the applications the technology is being used for. These technologies may be best investigated along with the standards from LS1 From Molecules to Organisms: Structure and Functions, as the students are learning about DNA and protein. Comparative DNA and protein sequence analysis is also a large part of current phylogenic research and could accompany LS4 Biological Change: Unity and Diversity. Students may also consider the pros and cons of biotechnical applications. Investigation of techniques utilized could come from field trips to witness the use of these techniques in practice, lab exercises, virtual labs, simulation activities, interviews with professionals, etc. Molecular techniques could include: PCR, electrophoresis, restriction enzyme digestion of DNA, DNA sequencing, plasmid-based transformation, transfection, etc. These techniques are used in fields of medicine, agriculture, biomedical engineering, forensic science, etc.

Karyotyping

Lampbrush Chromosomes

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Polytene Chromosomes

Polytene chromosomes are giant interphase chromosomes with several DNA strands placed side by side. They were discovered in the year 1881 by Balbiani in ...

Euchromatin

The extent of chromatin compaction can be studied by staining chromatin using specific DNA binding dyes. Under the microscope, the dense-compacted regions ...

Heterochromatin

X and Y Chromosomes

Among mammals, the gender of an organism is determined by the sex chromosomes. Humans have two sex chromosomes, X and Y. Every human diploid cell has 22 ...

The Y Chromosome Determines Maleness

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The Ratio of X Chromosome to Autosomes

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Investigate means by which karyotypes are utilized in diagnostic medicine.
EXPLANATION: Engineers incorporate growing scientific knowledge in order to increase benefits to humans and decrease cost and risks. Karyotyping technology and its use in medical diagnosis can complement the standards from LS3: Hereditary: Inheritance, and Variation of Traits, as the students are learning about chromosomal organization of genomic information that determines one's phenotype. Analyze a large set of karyotypes to identify common patterns. Identify variations from the most commonly observed patterns and correlate them with patient phenotypes to propose a cause and effect relationship, discussing limitations of correlative data interpretation.

BIO1.ETS2.2

Whole Body Regeneration

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stem-cell-therapy-for-tissue-regeneration

Stem Cell Therapy for Tissue Regeneration

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Introduction to Nuclear Reprogramming

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Methods of Nuclear Reprogramming

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Cloning of Dolly the Sheep

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Analyze scientific and ethical arguments to support the pros and cons of applications of a specific biotechnology technique such as stem cell usage, in vitro fertilization, or genetically modified organisms.
EXPLANATION: The utilization of new technologies in any field of science is dependent on both economic and social factors. In addition to evaluating these factors, scientists must also consider long-term consequences that may not be initially apparent. The emphasis should be on the construction of a rationale argument that supports a position on the use of an application with ethical and social impact. Students should begin to appreciate the differences in ethical values that exist, and recognize that discussion of these values is imperative as knowledge and technology continue to advance, even when resolutions of differences can be rare. After investigation of a specific biotechnology application, students can write a position paper and/or participate in a classroom debate.

BIO1.ETS2.3

ISH Probes for DNA and RNA Detection

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