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 DEPC and RNA: What It Is and Why It Matters

If you’ve ever worked with RNA, you know how notoriously unstable it can be. Unlike DNA, RNA is rapidly degraded by RNases—enzymes that are everywhere: on your hands, in the air, on your bench. To protect your precious RNA samples, one tool many scientists turn to is DEPC, or diethyl pyrocarbonate.

In this post, we’ll break down what DEPC is, why it’s used, how to store RNA safely, and how to handle DEPC properly in your experiments.

What Is DEPC?

DEPC (diethyl pyrocarbonate) is a chemical reagent used to inactivate RNases. It works by modifying certain amino acids—especially histidine—found in RNase enzymes. This modification renders the enzymes inactive, making your working solutions safer for RNA.

Common Uses of DEPC

DEPC is most often used to create RNase-free water, which is essential when preparing buffers, reagents, or working solutions for RNA extraction, purification, or analysis.

Some common applications include:

• Treating distilled water to make DEPC-treated water

• Decontaminating buffers (excluding those with incompatible components)

• Inactivating RNases on glassware or plasticware (though baking is often preferred for glassware)

⚠️ Important Handling Notes

Before you use DEPC, keep these key safety and compatibility tips in mind:

• Toxicity: DEPC is toxic and must be handled in a fume hood with gloves.

• Incompatible buffers: DEPC reacts with primary amines, including Tris, and should not be added to such solutions.

• Autoclaving: Always autoclave DEPC-treated solutions to inactivate any residual DEPC, converting it to ethanol and CO₂.

How to Make DEPC-Treated Water

Here’s a quick guide to preparing RNase-free water:

Materials:

• Distilled or Milli-Q water

• DEPC (100% stock solution)

• Autoclave-safe bottles

Steps:

1. Add 1 mL of DEPC per liter of water (final concentration 0.1% v/v).

2. Cap loosely and shake or stir thoroughly.

3. Let sit overnight at room temperature.

4. Autoclave the next day to remove residual DEPC.

Label clearly: DEPC-treated RNase-free water.

NB: if you do not want to prepare you own RNase-free water, you can buy certified nuclease-free water from various vendors.

When working with RNA in non-disposable plasticware, you may treat the plasticware with a 0.1M NaOH / 1mM EDTA, then rinse thoroughly with RNase-free water.

Safe Storage of RNA: Best Practices

Even with RNase-free reagents and tools, RNA can still degrade if not properly stored. RNases are endogenous (originate from the sample). Follow these best practices to protect your RNA samples:

For Biological Samples:

• Flash-freeze immediately using liquid nitrogen, or

• Add RNA stabilization reagents (e.g., RNAlater®) at the time of collection. Or if working with TRIzol as lysis reagent, add required amount and pipette up-and-down to lyse, before storing at -80oC.

• If processing right away, keep samples on ice and work quickly.

• For short-term storage, keep samples frozen or in stabilization reagents (some can preserve RNA at room temp for hours to days).

• For long-term storage, use –80°C with a stabilization reagent for best results and added protection during thawing.

For Purified RNA:

• Aliquot purified RNA into small RNase-free tubes to prevent repeated freeze-thaw cycles.

• Store in RNase-free water or TE buffer.

• –20°C: Short-term (up to a few weeks)

• –70°C or –80°C: Long-term

• Use tightly sealed tubes to prevent moisture and contamination.

• Monitor freezer temperatures regularly to ensure consistency.

These precautions help preserve RNA integrity, especially when preparing for sensitive downstream applications like RT-PCR, RNA-Seq, or Northern blotting.

Conclusion

RNA work demands meticulous technique, and DEPC is a powerful tool to keep RNases at bay. Combine DEPC-treated water with safe handling, quick processing, and proper storage to keep your RNA samples in top shape.

Bibliography

1. Agilent Technologies. (2004). RNA integrity assessment: RIN and its implications for RNA sample quality. Retrieved from https://www.agilent.com/cs/library/applications/5989-1165EN.pdf

2. QIAGEN. (n.d.). How should I store RNA samples? Retrieved from https://www.qiagen.com/us/knowledge-and-support/knowledge-hub/bench-guide/rna/introduction/general-remarks-on-rna-handling-storage-and-stabilization

3. Thermo Fisher Scientific. (n.d.). Guidelines for working with RNA. Retrieved from https://assets.thermofisher.com/TFS-Assets/LSG/manuals/fm_1910.pdf

June 02, 2025 Tags : Cloning , molecular biology , PCR

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