In-situ hybridization: Technique to detect mRNA localization || application of situ hybridization

Summary

This video explains the technique of in situ hybridization, which is used to detect and localize mRNA within tissue samples. It highlights the process of probing mRNA using complementary DNA sequences, followed by enzyme-linked or fluorescent detection methods. The video also emphasizes the technique’s application in studying mRNA localization in the hippocampus of the mouse brain. While effective for locating mRNA, the method cannot quantify its amount, and for precise quantification, real-time PCR is recommended. This method is widely used in developmental and cell biology to understand gene expression and localization.

Takeaways

• 😀 In situ hybridization is a technique to detect and localize specific mRNA in tissues.
• 😀 This technique helps answer two main questions: whether a specific mRNA is present and where it is located.
• 😀 The mouse brain hippocampus was used as an example to show how mRNA can be localized.
• 😀 The technique involves using a complementary DNA probe to hybridize with the mRNA of interest.
• 😀 After hybridization, probes are detected using enzyme-linked antibodies or fluorescently labeled antibodies.
• 😀 The enzyme-linked detection method involves alkaline phosphatase, which reacts with a substrate to produce a color change.
• 😀 The color reaction allows researchers to identify the exact location of mRNA expression in tissue samples.
• 😀 Fluorescently labeled probes allow for the technique to be modified into fluorescence in situ hybridization (FISH).
• 😀 In situ hybridization gives qualitative results but cannot quantitatively measure the number of mRNA copies present.
• 😀 For quantitative data, techniques like quantitative real-time PCR are necessary.
• 😀 Despite its limitations, in situ hybridization is widely used in developmental and cell biology for mRNA localization studies.

Q & A

What is the main purpose of in situ hybridization?

In situ hybridization is used to probe mRNA in tissues to determine whether a specific mRNA is present and where it is localized within a particular tissue or cell.

What are the two main questions in situ hybridization helps to answer?

In situ hybridization helps answer two key questions: 1) Whether a specific mRNA is present or not, and 2) The localization of that mRNA within the tissue.

What does the example in the video focus on in terms of mRNA localization?

The example in the video focuses on probing a particular mRNA in the hippocampus, specifically in the dentate gyrus region of the mouse brain.

What is the role of the probe in in situ hybridization?

The probe in in situ hybridization is a complementary DNA sequence that binds to the specific mRNA of interest, allowing for detection of the mRNA's presence and localization.

How is the probe detected in in situ hybridization?

The probe is detected using a label attached to it, which can be detected by either an enzyme-linked antibody system or a fluorescently labeled antibody system.

What is the function of the enzyme-linked antibody system in in situ hybridization?

The enzyme-linked antibody system binds to the labeled probe. The enzyme (e.g., alkaline phosphatase) then reacts with a substrate to produce a color reaction, which marks the presence of the mRNA in the tissue.

What is the color reaction used to visualize the mRNA in in situ hybridization?

The color reaction is based on the conversion of a substrate, BCIP, by alkaline phosphatase into a colored dye (formazan), which is visible as a blue or purplish color at the site of mRNA presence.

What are the main differences between traditional in situ hybridization and fluorescence in situ hybridization (FISH)?

Traditional in situ hybridization uses enzyme-linked antibody detection, while fluorescence in situ hybridization (FISH) uses fluorescently labeled antibodies to detect the probe through fluorescence microscopy.

What is the limitation of in situ hybridization in terms of quantifying mRNA?

In situ hybridization can show the localization and presence of mRNA, but it cannot quantitatively determine the exact number of mRNA copies. For quantitative estimates, techniques like quantitative real-time PCR are needed.

Why is in situ hybridization a valuable technique in developmental and cell biology?

In situ hybridization is valuable in developmental and cell biology because it allows researchers to study the spatial and temporal expression of genes within tissues, providing insights into gene function and regulation in specific anatomical regions.