RNA removal by double-RNase digestion
Introduction
DNA quantification by absorbance at 260 nm with a spectrophotometer is fast and easy, but may be lessaccurate than fluorescent quantification with dyes like SYBR® Green I1 or PicoGreen® (Molecular Probes).
The reason is that RNA is co-purified with DNA and absorbed at 260 nm. This may lead to an over-estimationof the amount of DNA. This protocol describes the use of double-RNase digestion to remove the RNA in
Oragene/saliva samples. After this RNase treatment, the DNA samples will give similar quantification resultsby absorbance or fluorescence.

Double-RNase digestion
This protocol uses two ribonucleases for double-digestion of RNA because treatment with Ribonuclease A
alone is not sufficient to degrade RNA into alcohol-soluble fragments. This is because Ribonuclease A cleaves
only at U- and C-nucleotides, leaving fragments large enough to be precipitated with alcohol. By including
Ribonuclease T1 (which cleaves at G-nucleotides), RNA can be digested into very small fragments that are
not precipitated by alcohol.

Equipment and reagents••••••

Ribonuclease A (Stock concentration: 1 mg/mL) (e.g., Sigma-Aldrich, Cat. No. R4875)
Ribonuclease T1 (Stock concentration: 6,000 units/mL) (e.g., Sigma-Aldrich, Cat. No. R1003)
Sodium chloride (NaCl) (Stock concentration: 5 M)
Ethanol (95% to 100%) at room temperature
TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) or other standard buffer
Microcentrifuge capable of running at 13,000 × gMethod
1. Purify a 500 μL aliquot of the Oragene® saliva sample† according to the prepIT™•L2P Laboratory protocolfor manual purification of DNA from 0.5 mL of sample2.
2. Resuspend the purified DNA pellet in 500 μL of 1× TE.
3. Add 5 μL of Ribonuclease A (final concentration of 10 μg/mL), and also add 2 μL of Ribonuclease T1(final concentration of 25 units/mL).
4. Incubate at 37°C for 30 minutes.
5. Add 10 μL of NaCl (final concentration of 0.1 M), and also add 1,000 μL of 95% ethanol (two volumes).
6. Mix well and incubate at room temperature for 10 minutes.
7. Collect the precipitated DNA by centrifugation at room temperature for 2 minutes at 13,000 × g.
8. Discard the supernatant and redissolve the DNA pellet in 500 μL of 1× TE.

†Saliva samples were collected with Oragene®•DNA or Oragene®•DISCOVER.

(outside the USA)(in the USA)

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www.dnagenotek.com • support@dnagenotek.com

References
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DNA quantification using the Fluorescence/DNase (F/D) assay. Replaced with DNA quantification using SYBR Green I dyeand a micro-plate reader. DNA Genotek. PD-PR-075.
Laboratory protocol for manual purification of DNA from 0.5 mL of sample. DNA Genotek. PD-PR-006.

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Oragene®•DNA is not available for sale in the United States.
Oragene®•DISCOVER is for research use only, not for use in diagnostic procedures.
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All DNA Genotek protocols, white papers and application notes, are available in the support section of our website at www.dnagenotek.com.

PD-PR-040 Issue 3/2011-11© 2011 DNA Genotek Inc., a subsidiary of OraSure Technologies, Inc., all rights reserved.

www.dnagenotek.com • support@dnagenotek.com

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