Protocol – Preparing RNA free of DNA Using DNase I Digestion

Overview

• DNase I is an endonuclease that selectively cleaves phosphodiester bonds of both single- and double-stranded DNA without cleaving RNA. This property of DNase I is utilized to remove DNA contamination from the RNA samples.
• This method efficiently removes trace to moderate amounts of DNA (up to 10 µg/ml) from the RNA samples. 
• RNA preparations are briefly treated with DNase I at 37°C. 
• After incubation, DNase I must be inactivated by the addition of EDTA and heat treatment. If required, Phenol:Chloroform extraction can be performed to remove the DNase I from the RNA samples. 

Requirements

Equipment and disposables
> Water bath/Heat block (preset to 37ºC and 75ºC)
> Microcentrifuge tubes (1.5 mL) (RNase-free)
> Micropipette (P10, P100)
> Micropipette tips
> Microcentrifuge
>Deionized water, RNase-free

Reagents and solutions
> RNA sample
> DNase I enzyme (RNase-free)
> 10x DNase I reaction buffer (supplied with the DNase I enzyme)
> 50 mM EDTA (also called stop solution, supplied with the DNase I enzyme by some suppliers)

Procedure: 

Note:
♦ The procedure described here is a general instruction to remove DNA contamination using DNase I digestion. Always consider and read carefully the instruction manual provided with DNase I enzyme from different suppliers.
♦ This method is suitable for removing DNA contamination up to 10 µg/ml. If the RNA preparations contain more than 10 µg/ml DNA contamination, either dilute the sample or use other methods suitable to remove large amounts of DNA contamination from the RNA preparations such as LiCl precipitation.

Precaution
Maintain RNase-free conditions throughout the protocol. Use RNase-free glassware, and reagents.

Step 1: Set the reaction as follows….

Set this reaction in a 1.5 ml or 0.5 ml microcentrifuge tube. Mix the content by tapping the tube gently with your fingers. If necessary, give a short spin in a minicentrifuge to bring all the content to the bottom of the tube.

Note:
The total final volume of the reaction is 20 µl. This reaction volume is suitable to process up to 10 µg RNA. To add more RNA sample volume, reduce the volume of water appropriately. For example, if you want to add 5 µl of RNA sample, reduce the volume of water to 12 µl.

Tip:
Scale up the reaction and add reaction components appropriately if you want to add more than 10 µg of RNA (for example set 100 µl reaction to process 50 µg RNA). 

Step 2: Incubate at 37°C for 30 min.

Step 3: Heat inactivate DNase I enzyme after adding EDTA
◊ Add 1µL of EDTA (stock solution 50 mM) and incubate at 75°C for 5–10 min.
◊ Centrifuge the tube briefly to collect the content to the bottom of the tube and store it on ice.

Precautions:
RNA undergoes spontaneous degradation at high temperatures in the presence of divalent cations. EDTA, a chelator, protects the RNA from spontaneous degradation at high temperatures by sequestering divalent cations.

Result: DNA-free RNA is now ready for further manipulation such as quantitative PCR.