RAM-FISH: A New Way to Study RNA

Living things made up of many cells, like animals and plants, have complicated systems inside them. These systems are controlled by tiny molecules known as RNA. The way these RNA molecules show up in cells can tell us a lot about how tissues develop and how diseases start. Many scientists want to find easy and reliable ways to see multiple RNA molecules in tissues without messing things up too much.

To study these RNA molecules, scientists need methods that work well and don't take up too much time or effort. They also want to ensure they can look at different types of samples without a lot of preparation. Recent technology allows scientists to check RNA in single cells while keeping the tissue structure intact. There are two main methods: one that uses sequencing and another that uses colorful probes.

#RNA Detection Techniques

#Sequencing-based Methods

Sequencing-based methods involve taking apart and reading the RNA sequences to find out which genes are active. Popular techniques include:

• Visium: This method allows for spatial analysis of RNA in tissues, providing a map of gene activity.
• Stereo-seq: This technique works similarly but has a different approach to capture data.
• Slide-seq: This method captures RNA signals using a specific slide setup.

#Imaging-based Methods

Imaging-based methods use special fluorescent tags to mark where RNA is and how much there is. Some well-known techniques include:

• MERFISH: This method uses multiple rounds of imaging to collect data on many RNA molecules at once.
• osmFISH: It helps visualize RNA in a very detailed way.
• CosMx SMI: This innovative approach tracks RNA in tissue samples.
• STARmap: This technique gives high-resolution images of where RNA is located in tissues.
• seq-FISH: Another method that allows scientists to see RNA signals very clearly.
• FISH&CHIPS: This is a newer method that works alongside other techniques.

While these methods are helpful, they often come with issues like complicated preparation steps, high costs, and the risk of getting false signals.

#Improvements in RNA Localization Methods

Scientists have come up with better ways to amplify signals and suppress background noise. Methods like HCR3.0 and SABER-FISH have made it possible to see signals more clearly and reduce background interference. However, these techniques still have their own problems, such as:

1. Limited ability to handle many RNA targets at once.
2. Lengthy experimental timelines.
3. Labor-intensive protocols that need expert handling, which can lead to mistakes.

Some new methods, like cycleHCR and EASI-FISH, have improved the number of targets they can handle, but they require complicated setups and are not easy for all labs to use.

#RAM-FISH: A New Solution

Enter RAM-FISH! This method combines advanced techniques to efficiently detect more than 30 RNA targets all at once. It's quicker and simpler than older methods, making it easier for scientists to use. Previously, researchers have tested this method on butterfly scales and fish brains, and now it's been enhanced to allow for multiple rounds of detection.

#Workflow of RAM-FISH

The workflow for RAM-FISH is straightforward. It starts with the preparation of tissue samples, which can be done either manually or using automated systems. After gathering the tissues, scientists fix and permeabilize them. Then, they either do the testing themselves or let machines handle it.

The basic steps include:

1. Collecting Tissues: First, scientists take samples from the organism, whether it's a butterfly or a fish.
2. Fixing and Preparing: Tissues are treated to make them easier to work with and to keep the cells intact.
3. Probing the RNA: They then use special probes that bind to the RNA of interest. After that, they use additional probes to amplify the signals they want to see.
4. Imaging: Finally, the samples are examined under a special microscope to capture the RNA signals.

In the manual setup, the hybridization, washing, and signal removal steps happen mostly in glass plates or small tubes. The automated approach uses a fluidic system to streamline the process, making it more efficient.

#Probes Preparation

To prepare probes for detecting RNA, researchers created helpful Excel templates. These templates help design oligonucleotides that can attach to the specific RNA they want to study. They use a gene sequence from databases and prepare the probes to ensure they bind correctly.

#Practical Applications of RAM-FISH

#Butterfly Research

One exciting application of RAM-FISH is looking at developing butterfly wings. Butterflies have unique color patterns that change as they grow. Scientists have studied up to 33 genes in different stages of development to see how they behave.

For example:

• Wnt1, Wnt6, and Wnt10: These genes showed consistent patterns related to wing margins and spots, matching past studies.
• Cubitus interruptus (ci): This gene was found in certain areas of the wing, which aligns with previous work.

Using RAM-FISH allowed researchers to see how complex gene expression is during the butterfly’s development, helping to understand their growth and color patterns.

#Zebrafish Brain Studies

Zebrafish larvae are another great example of RAM-FISH in action. Because of their simple structure and transparency, zebrafish are ideal for studying how genes work in the brain during early stages of life. Researchers used RAM-FISH to check how certain genes express in their brains, which is important for understanding behaviors.

For instance, several genes related to nerve function were examined, showing where and how they are active. This helps build a clearer picture of brain function and development.

#Challenges and Robustness Analysis

While RAM-FISH is a powerful tool, scientists needed to address some challenges, like making sure signals stay strong through multiple cycles of detection. They looked specifically at the gene optomotor-blind (omb) to analyze how signals degrade over time.

To test the method's reliability, they compared images taken after different rounds of detection. They found that while there might be some loss of signal, the overall patterns remained clear, supporting the robustness of the RAM-FISH method.

#Conclusion

RAM-FISH represents an exciting advancement in the field of RNA detection and localization. It provides a simpler, faster, and more reliable way to study gene expression in various organisms. Whether it's unraveling the secrets of butterfly wing development or providing insights into zebrafish brains, this method has the potential to revolutionize how scientists explore the world of gene expression.

In the ever-changing landscape of scientific research, RAM-FISH holds promise for many researchers looking for efficient methods to unlock the fascinating world of RNA and its role in life's processes. With this tool in their arsenal, scientists are likely to make discoveries that will enhance our understanding of biology, development, and disease in ways we can only anticipate.

So let’s keep our eyes peeled; who knows what amazing findings await us with this new approach to RNA study!