Visualising mRNA distribution in tissues

Question 1

What is in situ hybridisation?

Answer 1

technique that allows the detection and visualization of specific nucleic acid sequences within a fixed tissue or cell sample. The technique uses a labeled probe, typically a small DNA or RNA fragment that is complementary to the target sequence of interest, to hybridize to the target in the sample. The probe is labeled with a detectable marker, such as a fluorescent dye, and once it binds to its complementary sequence, the presence and location of the target sequence can be visualized under a microscope.

Question 2

What is ISH used for?

Answer 2

to study gene expression, identify specific cell types within tissues, detect genetic aberrations, and study the spatial organization of DNA and RNA within cells and tissues.

Question 3

What are the steps of ISH?

Answer 3

1. Sample preparation: The tissue or cell sample of interest is first prepared by fixation, embedding, and sectioning to create thin slices for analysis.
2. Probe synthesis: A probe is designed to be complementary to the target sequence of interest. This can be a DNA or RNA probe, and it is often labeled with a detectable marker, such as a fluorescent dye.
3. Hybridization: The probe is added to the sample and allowed to hybridize to its complementary target sequence. The sample is then incubated under specific conditions to promote hybridization.
4. Washing: The sample is washed to remove any unbound probe, leaving only the probe that has specifically bound to its target sequence.
5. Detection: The labeled probe is detected using a specialized microscope or imaging system that is capable of visualizing the fluorescent or other signal emitted by the label.
6. Analysis: The results of the in situ hybridization can be analyzed and interpreted to provide insights into gene expression, cell types, genetic aberrations, or other features of the sample being studied.

Question 4

What is a probe?

Answer 4

A probe is a short DNA or RNA molecule that is complementary to a specific target sequence of interest. The probe is designed to hybridize or bind to the target sequence under specific conditions. When the probe binds to the target sequence, it can be used to detect the presence and location of the target sequence in a sample.

Question 5

What is a riboprobe?

Answer 5

A riboprobe is a type of probe used in molecular biology that is made from RNA instead of DNA. Riboprobes are synthesized in vitro using an RNA polymerase enzyme and a DNA template that contains a specific sequence of interest. The RNA produced by the polymerase is complementary to the DNA template, and it can be used to detect the presence and location of specific RNA molecules in a sample.

Question 6

how does a DIG labelled probe work?

Answer 6

A DIG (digoxigenin)-labeled probe is a type of nucleic acid probe that has been labeled with the DIG molecule, which is a steroid-like molecule derived from the foxglove plant. The DIG label is added to the probe during its synthesis, typically by incorporating DIG-labeled nucleotides into the probe sequence. During hybridization, the DIG-labeled probe binds to its complementary target sequence in the sample, and the DIG label can be detected using an antibody-based detection system. The detection system involves the use of an anti-DIG antibody that is conjugated to a detectable marker, such as a fluorescent dye or an enzyme. After hybridization, the sample is treated with the anti-DIG antibody, which specifically binds to the DIG label on the probe. The detectable marker on the antibody is then used to visualize and detect the presence and location of the probe in the sample.

Question 7

How is BCIP/NBT used in ISH?

Answer 7

Commonly used substrates in in situ hybridization (ISH) protocols for the detection of alkaline phosphatase (AP)-labeled probes. When AP-labeled probes hybridize to their target sequences, the AP enzyme cleaves the phosphate groups from the BCIP/NBT substrates, resulting in the formation of a blue-purple precipitate that can be visualized under a microscope.

The use of BCIP/NBT in ISH typically involves the following steps:

1. Fixation: The sample (usually cells or tissue sections) is fixed using a suitable fixative, such as paraformaldehyde or formalin, to preserve the integrity of the cells and prevent RNA degradation.
2. Prehybridization: The sample is treated with a prehybridization solution to block non-specific binding sites and reduce background staining.
3. Hybridization: The AP-labeled probe is added to the sample and allowed to hybridize to its complementary target sequence. The hybridization conditions (temperature, time, and buffer composition) are optimized to ensure specific and efficient hybridization of the probe to the target sequence.
4. Post-hybridization washes: The sample is washed to remove unbound probe and reduce background staining.
5. Detection: The BCIP/NBT substrate solution is added to the sample, and the AP enzyme cleaves the phosphate groups from the substrate to produce a blue-purple precipitate. The reaction is stopped by washing the sample with a suitable stop solution.
6. Counterstaining: A counterstain, such as nuclear fast red or eosin, may be added to the sample to enhance the visualization of the target sequence.
7. Mounting: The sample is mounted onto a glass slide and coverslipped for microscopy.

The intensity and distribution of the blue-purple precipitate are proportional to the amount and localization of the target sequence present in the sample. Therefore, the BCIP/NBT reaction can be used to detect and localize specific RNA sequences in cells and tissues.

Question 8

What is the name of the molecular tag in the probe recognised by the secondary antibody?

Answer 8

Depends on the labeling method used for the probe.
If the probe is labeled with a hapten, such as biotin or digoxigenin (DIG), then the secondary antibody used in detection is usually conjugated to an enzyme or a fluorescent dye. The enzyme or fluorescent dye is the molecular tag that is recognized by the secondary antibody. For example, if the probe is labeled with biotin, the secondary antibody may be conjugated to an enzyme such as horseradish peroxidase (HRP) or alkaline phosphatase (AP), which can catalyze a colorimetric or fluorescent reaction for visualization.

Question 9

What is the the general protocol for riboprobe preparation?

Answer 9

1. Design the probe sequence: Choose a target RNA sequence of interest and design a complementary DNA (cDNA) sequence to be used as the probe. The cDNA can be generated by reverse transcription of RNA using a reverse transcriptase enzyme and specific primers.
2. Clone the probe into a vector: Insert the cDNA sequence into a vector that contains a promoter for RNA polymerase. The most commonly used vectors for generating riboprobes are the pBluescript or pGEM-T vectors.
3. Linearize the vector: Use a restriction enzyme to linearize the vector downstream of the probe sequence. This will allow the RNA polymerase to transcribe the probe sequence.
4. Transcribe the probe: Use a DNA-dependent RNA polymerase (e.g. T7, T3 or SP6) to transcribe the probe from the linearized vector in the presence of labeled nucleotides (e.g. [α-32P]-UTP, DIG-UTP or fluorescent-labeled UTP). The type of labeled nucleotide used will depend on the detection method chosen.
5. Purify the probe: Purify the riboprobe using a purification kit or a combination of column chromatography, gel electrophoresis, and ethanol precipitation. This will remove unincorporated labeled nucleotides and other impurities that can interfere with hybridization.
6. Quantify the probe: Measure the concentration of the riboprobe and the specific activity of the label using a scintillation counter or a spectrophotometer.