Tips for Handling and Storing Tissue Samples Prior to DNA Extraction

Summary

This video script emphasizes the critical steps for preserving DNA integrity during extraction. It suggests using stabilizing reagents like RNAlater or flash freezing with liquid nitrogen or dry ice to prevent degradation by cellular nucleases. Tissue samples, especially from metabolically active organs, should be stored at -80°C. The script also discusses the benefits of using chemically stabilized tissue and the techniques for creating tissue powder for easier DNA extraction, highlighting the importance of immediate use and avoiding re-freezing of thawed samples.

Takeaways

• 🧬 **Preservation is Key**: Proper handling and storage are crucial for maintaining DNA integrity and preventing extraction failures.
• ❄️ **Immediate Preservation**: Tissue samples should be preserved right after harvest to avoid DNA degradation by cellular nucleases.
• 🔥 **Metabolically-Active Tissues**: Tissues like liver, kidney, pancreas, and intestine require immediate preservation due to high nuclease content.
• 🧪 **Stabilizing Reagents**: Use of reagents like RNAlater or flash freezing with liquid nitrogen or dry ice is recommended for sample preservation.
• ❄️ **Storage Conditions**: Samples should be stored at -80°C to maintain their integrity until DNA extraction.
• 🧪 **Chemical Stabilization**: NEB prefers chemically stabilized tissue for convenient and stress-free DNA purification.
• 🧊 **Freezing as an Alternative**: When stabilization reagents are not available, freezing samples is an effective preservation method.
• 🌨️ **Tissue Powder Advantages**: Storing tissues as powder provides ease of use, homogeneity, faster lysis, and higher molecular weight DNA.
• ⚒️ **Preparation of Tissue Powder**: Tissue powder is made by shock freezing in liquid nitrogen and grinding with a mortar and pestle.
• 🔪 **Tissue Pieces**: When making a powder is not possible, cutting samples into small pieces is advised, with immediate use after thawing to prevent nuclease activity.

Q & A

Why is it crucial to preserve the integrity of DNA during extraction?

Preserving the integrity of DNA is crucial because improper handling and storage can lead to DNA extraction failures, affecting the quality, purity, and yield of the extracted DNA.

What are the recommended methods for preserving tissue samples to prevent DNA degradation?

Tissue samples should be preserved using a stabilizing reagent like RNAlater or by flash freezing with liquid nitrogen or dry ice to prevent DNA degradation by cellular nucleases.

Why is it important to store samples at minus 80°C after preservation?

Storing samples at minus 80°C ensures that the DNA remains stable and prevents further degradation until the time of extraction.

What are the advantages of using chemical stabilization reagents like RNAlater for preserving tissue samples?

Chemical stabilization reagents protect against nuclease activity, allowing for convenient handling, transport, and storage at room temperature without the need for freezing.

How does chemical stabilization affect the handling of tissue samples during DNA extraction?

Chemically stabilized samples maintain their shape and rigidity when thawed, making them easy to cut and handle, which simplifies the DNA extraction process.

What are the guidelines for freezing tissue samples when stabilization reagents are not used?

When not using stabilization reagents, samples can be stored as pieces or powder. It's recommended to store tissues as a powder for ease of dispensing and higher DNA yield, but this requires working with liquid nitrogen.

Why is tissue powder considered a better option for DNA extraction compared to tissue pieces?

Tissue powder provides a homogeneous representation of the sample, is faster to lyse, and typically yields higher molecular weight DNA compared to tissue pieces.

What is the recommended procedure for creating tissue powder for DNA extraction?

Tissue powder is made by shock freezing samples in liquid nitrogen and then grinding them with a mortar and pestle before transferring to tubes for storage.

What should be considered when storing tissue samples as pieces instead of powder?

When storing as pieces, it's important to cut samples into the smallest possible pieces to facilitate lysis. Thawed samples should be used immediately to prevent nuclease activity, and tissue pieces should not be refrozen.

How does the size of tissue pieces affect the DNA extraction process?

Smaller tissue pieces are easier to lyse and may result in a higher average DNA fragment length compared to larger pieces, which can be degraded by nucleases during the lysis process.

What should one do if they have questions regarding DNA extraction procedures?

If there are questions about DNA extraction, scientists at NEB are available for assistance and can be contacted at [email protected].

Outlines

00:00

🧬 DNA Preservation for Quality Extraction

This paragraph emphasizes the importance of preserving DNA integrity through proper handling and storage to prevent extraction failures. It advises using stabilizing reagents like RNAlater or flash freezing with liquid nitrogen or dry ice to prevent DNA degradation by cellular nucleases, especially in metabolically-active tissues. The paragraph also covers the benefits of chemical stabilization for convenience and protection against nuclease activity, allowing for room temperature handling and storage. Additionally, it discusses the advantages of storing tissue as a powder for easier dispensing and higher DNA yield, and the importance of using tissue pieces promptly once thawed to avoid nuclease reactivation.

Keywords

💡DNA Integrity

DNA Integrity refers to the condition of DNA being unaltered and undamaged, which is crucial for accurate genetic analysis. In the context of the video, maintaining DNA integrity is the primary goal to ensure successful DNA extraction. The script emphasizes the importance of proper handling and storage to prevent DNA degradation by cellular nucleases, especially in metabolically-active tissues.

💡DNA Extraction

DNA Extraction is the process of isolating DNA from biological samples such as tissues. The video script discusses various methods to ensure that DNA extraction yields high-quality DNA. It is central to the video's theme as it directly relates to the techniques for preserving sample integrity and achieving maximal purity and yield.

💡Tissue Samples

Tissue Samples are biological specimens taken from an organism, used for various laboratory analyses, including DNA extraction. The script mentions that tissue samples should be preserved immediately after harvest to prevent DNA degradation, highlighting the significance of timely and proper sample handling in the context of DNA extraction.

💡Cellular Nucleases

Cellular Nucleases are enzymes within cells that break down nucleic acids, such as DNA. In the video, it is mentioned that improper handling can lead to DNA degradation by cellular nucleases, particularly in tissues with high nuclease content like liver, kidney, pancreas, and intestine. This term is key to understanding the necessity of preservation methods discussed.

💡RNAlater

RNAlater is a commercial stabilizing reagent used to preserve the integrity of RNA and DNA in biological samples. The script recommends using RNAlater or similar reagents for preserving tissue samples, which allows for convenient handling and storage at room temperature and protects against nuclease activity.

💡Flash Freezing

Flash Freezing is a rapid freezing technique that preserves the structure of biological samples by minimizing ice crystal formation. The video script suggests flash freezing as a method to preserve tissue samples, either with liquid nitrogen or dry ice, to prevent DNA degradation before extraction.

💡Minus 80°C Storage

Minus 80°C Storage refers to the practice of storing biological samples at ultra-low temperatures to preserve their integrity. The script specifies that samples should be stored at -80°C until ready for DNA extraction, indicating the importance of cold storage in maintaining sample quality.

💡Tissue Powder

Tissue Powder is a form of biological sample prepared by grinding frozen tissue into a fine powder. The script recommends storing tissues as a powder for ease of dispensing, aliquoting, and higher DNA yield. It also mentions that tissue powder provides a homogeneous representation of the sample and is faster to lyse.

💡Liquid Nitrogen

Liquid Nitrogen is a cryogenic liquid used to achieve extremely low temperatures for preserving biological samples. In the context of the video, liquid nitrogen is used for flash freezing samples to create tissue powder, emphasizing its role in the preservation process for DNA extraction.

💡Lysis

Lysis is the process of breaking open cells to release their contents, such as DNA. The script discusses the ease of lysing tissue powder compared to tissue pieces, and how this can affect the speed of DNA extraction and the quality of the DNA obtained, making lysis a critical step in the DNA extraction process.

💡Nuclease Activity

Nuclease Activity refers to the enzymatic action of nucleases that can degrade nucleic acids. The video script warns against the reactivation of nucleases in thawed samples, which can lead to DNA degradation. This term is crucial for understanding the importance of immediate use of thawed samples and the avoidance of re-freezing.

Highlights

Proper handling and storage are crucial for preserving DNA integrity.

Improper handling is a common cause of DNA extraction failures.

Tips will be shared to ensure sample integrity for maximal purity and yield.

Tissue samples should be preserved immediately after harvest to prevent DNA degradation.

Metabolically-active tissues like liver, kidney, pancreas, and intestine have high nuclease content.

Preservation using stabilizing reagents like RNAlater or flash freezing is recommended.

Samples should be stored at -80°C until DNA extraction.

NEB prefers chemically stabilized tissue for convenient purification.

Chemically stabilized samples are protected against nuclease activity.

Stabilized tissues maintain shape and rigidity for easy handling.

Freezing samples is an alternative preservation technique.

Frozen samples can be stored as pieces or powder for DNA extraction.

Tissue powder provides a homogeneous representation and higher molecular weight DNA.

Tissue powder requires liquid nitrogen and must be kept on dry ice during preparation.

Tissue pieces should be cut into small pieces for faster lysis and to minimize nuclease exposure.

Thawed samples should be used immediately to avoid nuclease activity.

NEB scientists are ready to help with any questions regarding DNA extraction.

Transcripts

In order to reliably extract high quality DNA, it is important to preserve the integrity

of the DNA with proper handling and storage.

Improper handling and storage is one of the most common reasons for DNA extraction failures.

In the following video, we’ll share some tips that will ensure that sample integrity

is maintained so that you can achieve maximal purity and yield.

Immediately after harvest, tissue samples should be preserved to prevent DNA degradation

by cellular nucleases.

This is especially important for metabolically-active tissues such as liver, kidney, pancreas, and

intestine, which all have high nuclease content.

We recommend preservation using a stabilizing reagent such as RNAlater or by flash freezing

the sample, either with liquid nitrogen or dry ice.

Samples should then be stored at minus 80°C until you are ready to extract the DNA.

First, we will discuss using stabilization reagents such as RNAlater to preserve tissue

samples.

At NEB, our preference is to work with chemically stabilized tissue because it allows for a

more convenient and stress-free purification experience.

Chemically stabilized samples are protected against nuclease activity, which allows for

handling, transport and storage at room temperature and under less stringent conditions than when

working with frozen samples.

Stabilized tissues also maintain their shape and rigidity when thawed, which allows them

to be easily cut and handled.

When stabilization reagents are not available or when users prefer not to use them, freezing

your samples is another preservation technique.

Next, we will provide some guidelines for freezing your samples.

You can store frozen samples as pieces or as a powder.

If possible, we recommend storing tissues as a powder.

Tissue powder can easily be dispensed and aliquoted, it provides a homogeneous representation

of the sample, it is faster to lyse, and it typically produces higher molecular weight

DNA.

However, it does require working with liquid nitrogen, and, as powder thaws quickly, it

must be kept on dry ice during your prep.

Samples stored as powders are shock frozen in liquid nitrogen and then ground with a

mortar and pestle.

The powder can then easily be transferred to tubes for storage.

In some cases, like when working with small tissue samples, making a powder is not possible.

In other cases, users may prefer to work with tissue pieces.

When doing so, it’s important to cut samples into the smallest pieces possible.

Thawed samples should be used immediately, as nucleases can become active, and you should

never re-freeze tissue pieces.

Tissue pieces also take longer to lyse and may have a slightly lower average fragment

length because exposure to nucleases can cause degradation while the tissue is being lysed.

We hope that these tips have been helpful.

If you have any questions, our scientists are ready to help.

Contact us at info at NEB.com.