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].