DNase Treatment in RNA Extraction: Removing DNA Contamination for Pure RNA Samples

 DNase Treatment in RNA Extraction: A Guide to Removing DNA Contamination

When working with RNA for downstream applications like RT-PCR, RNA-seq, or transcriptome analysis, DNA contamination can cause misleading results and false positives. This is where DNase treatment comes in — an essential step that ensures your RNA prep is free from genomic DNA. In this blog, we’ll explore why DNase treatment is important, and give you a typical protocol synthesised from literature, manufacturer recommendations, and lab best practices.

Why DNase Treatment Is Necessary

During RNA extraction, it’s almost impossible to avoid co-purifying some genomic DNA, especially from samples with high cell or tissue content. This contamination can:

• Amplify in minus-RT controls (false positives)

• Distort gene expression quantification in qPCR

• Add unwanted reads in RNA-seq

DNase enzymes specifically degrade DNA without harming RNA when used under RNase-free conditions, making them a go-to step for high-quality RNA prep.

Types of DNase Treatment

1. On-column digestion

• Performed during RNA purification using silica spin columns.

• DNase I is applied directly to the membrane after RNA binding.

• Convenient and integrates into the workflow.

• Example: Qiagen RNase-Free DNase Set (15 minutes at 20–30°C with Buffer RDD).

2. In-solution digestion

• Performed after RNA is already purified.

• Offers more control and flexibility for challenging samples.

• Example: TURBO DNase (Ambion/Thermo Fisher) for high-activity digestion.

Example DNase Protocol (In-solution)

Materials:

• RNase-free DNase I or TURBO (engineered) DNase

• Supplied DNase reaction buffer

• RNase-free water and tubes

• EDTA (0.5 M) for inactivation (if using heat method)

• Cleanup method (column kit or phenol:chloroform + ethanol precipitation)

Steps:

1. Place RNA (≤10 µg) in RNase-free water, total volume 10–20 µL.

2. Add DNase buffer to the final recommended concentration.

3. Add DNase enzyme:

• TURBO DNase: ~1 U per µg RNA, incubate at 37°C for 30 min.

• Wild-type DNase I: ~1 U per µg RNA, incubate at 37°C for 10–30 min.

4. Gently mix by inversion (avoid vortexing).

5. Inactivate and remove DNase:

• EDTA to 15 mM + heat at 75°C for 10 min (fast, but may leave buffer salts)

• Phenol:chloroform extraction + ethanol precipitation (very clean)

• Column cleanup (fast and effective; removes DNase and salts)

On-column DNase Protocol (Example)

1. After RNA binds to column, apply DNase I mix (usually DNase + Buffer RDD).

2. Incubate 15 min at room temp (20–30°C).

3. Continue with wash steps as per the kit’s instructions.

4. Elute RNA as usual.

Verifying DNase Success

• Minus-RT control in RT-qPCR: No amplification means effective DNA removal.

• Genomic target qPCR: Target an intron or DNA-only sequence to check for contamination.

• For RNA-seq: rRNA depletion or poly(A) selection also helps reduce DNA read-through.

Tips and Common Pitfalls

• Always work RNase-free — RNase contamination will destroy your RNA faster than DNase works on DNA.

• TURBO DNase is more active and can remove stubborn contamination in difficult samples.

• Don’t skip the cleanup step — even inactivated DNase can interfere with downstream enzymes.

• Avoid vigorous mixing that might shear RNA.

Conclusion

DNase treatment is a small step that makes a big difference in RNA quality. It can be performed via  an on-column method for convenience or an in-solution approach can be used.

References

1. Thermo Fisher Scientific. (n.d.-a). TURBO™ DNase product information sheet (Pub. No. 4393900 Rev. B) [PDF]. Retrieved from Thermo Fisher Scientific website

2. Thermo Fisher Scientific. (n.d.-b). TURBO DNA-free™ kit user guide (Pub. No. 1907M) [PDF]. Retrieved from Thermo Fisher Scientific website

3. Thermo Fisher Scientific. (n.d.-c). DNA-free™ kit user guide (Invitrogen™, Pub. No. 1906M Rev. F) [PDF]. Retrieved from Thermo Fisher Scientific website

4. Thermo Fisher Scientific. (n.d.-d). The world’s best DNase – Tech note. Retrieved from Thermo Fisher Scientific website

5. QIAGEN. (n.d.-a). RNase-Free DNase set [Product information]. Retrieved from QIAGEN website

6. QIAGEN. (n.d.-b). RNase-Free DNase set product sheet [PDF]. Retrieved from QIAGEN website

7. QIAGEN. (2023). RNeasy® Mini handbook [PDF]. Retrieved from QIAGEN website

8. QIAGEN. (2023). RNeasy 96 handbook [PDF]. Retrieved from QIAGEN website

August 11, 2025 Tags : DNase treatment , molecular biology protocol , remove DNA contamination , RNA extraction

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