Northern blotting technique

Summary

This video tutorial from Shos Biology explores Northern blotting, a technique for detecting specific RNA sequences within a cell. It emphasizes the similarity between Northern and Southern blotting, explaining that the former focuses on RNA while the latter targets DNA. The process involves RNA extraction, denaturing to linearize the molecules, separation using agarose gel electrophoresis, and transfer onto a membrane for hybridization with a complementary DNA probe. The technique is crucial for identifying specific RNA fragments, and the video promises further insights into related blotting methods.

Takeaways

• 📚 Northern blotting is a technique used to detect specific RNA sequences within a mixture of RNA.
• 🔍 It is similar to Southern blotting, which detects DNA, and both involve hybridization techniques.
• 🕵️‍♂️ The name 'Northern blotting' was derived from 'Southern blotting' and does not relate to geographical directions.
• 🧬 The process involves three main stages: extraction of RNA, separation using denaturing gel electrophoresis, and hybridization with a complementary DNA probe.
• 🧪 RNA is extracted from the cell and then broken down into smaller fragments to eliminate complex secondary structures.
• 🔬 Denaturing gel electrophoresis is used to resolve the secondary structure of RNA into a linear form for accurate separation based on length.
• 🧴 Formaldehyde is added to the gel to help resolve the secondary structure of RNA into a linear form for proper separation.
• 📝 Northern blotting uses a different type of membrane for transferring RNA from the gel compared to Southern blotting, often using aminooxy methy filter paper for better RNA binding.
• 💧 The transfer of RNA from the gel to the membrane is facilitated by capillary action, with the setup including a buffer, sponge, gel, membrane, and paper towels.
• 🔦 A radioactive or chemiluminescent probe is used to bind to the specific target RNA, allowing for the detection and identification of the RNA of interest.
• 📉 The final step involves developing an autoradiograph or using a detection method that reveals the location of the target RNA within the membrane.

Q & A

What is the main purpose of Northern blotting?

Northern blotting is a technique used to detect specific RNA sequences within a mixture of RNA extracted from cells.

What is the relationship between Southern blotting and Northern blotting?

Southern blotting and Northern blotting are similar techniques, with the main difference being that Southern blotting is used for detecting DNA, while Northern blotting is used for detecting RNA.

Who discovered the Southern blotting technique?

The Southern blotting technique was discovered by Edwin Southern in 1975.

What is the significance of using formaldehyde in the gel during Northern blotting?

Formaldehyde is used in the gel to help resolve the secondary structure of RNA into a linear form, which is necessary for accurate separation and detection of RNA fragments.

Why is aminooxy methy paper preferred over nitrocellulose paper in Northern blotting?

Aminooxy methy paper has a greater binding affinity towards RNA compared to nitrocellulose paper, making it a better medium for transferring and detecting RNA during Northern blotting.

What is the role of the probe in Northern blotting?

The probe in Northern blotting is a complementary DNA strand that binds specifically to the target RNA sequence. It is often labeled with a radioactive or chemiluminescent molecule to facilitate the detection of the target RNA.

How does the denaturing gel electrophoresis process help in Northern blotting?

Denaturing gel electrophoresis helps to break down the complex secondary structures of RNA into linear forms, allowing for the separation of RNA fragments based on their length.

What is the purpose of the buffer in the blotting setup?

The buffer in the blotting setup facilitates the capillary action that is necessary for transferring the RNA from the gel to the membrane or filter paper.

Why is the RNA transferred from the gel to a membrane or filter paper?

The RNA is transferred to a membrane or filter paper because agarose gel is fragile and not suitable for the probing process, which requires the application of various solutions and buffers.

What is the final step in detecting the target RNA after the transfer to the membrane?

The final step involves adding the probe to the membrane, which binds specifically to the target RNA. The detection is then achieved through the signal given by the radioactive or chemiluminescent label on the probe.

Why is DNA typically used as a probe in Northern blotting instead of RNA?

DNA is typically used as a probe in Northern blotting because it has better interaction with RNA and is more stable than RNA, which can easily degrade. The DNA probe is designed based on the complementary RNA structure.