DNA isolation is a foundational technique in molecular biology, but extracting DNA from a biological sample often results in the co-purification of RNA. Structurally, RNA is very similar to DNA, making separation challenging using standard chemical extraction methods. RNA is typically present in vast excess within cells, meaning a significant amount carries over into the final DNA preparation. This contamination interferes with many subsequent molecular analyses. This article details established methods to effectively remove RNA and ensure the resulting DNA sample is pure for reliable downstream applications.
Why RNA Contamination Interferes with DNA Analysis
Residual RNA primarily causes the overestimation of actual DNA concentration during standard spectrophotometric quantification. Both DNA and RNA absorb ultraviolet light strongly at 260 nanometers (nm), meaning a spectrophotometer cannot distinguish between the two molecules. The resulting inflated concentration measurement causes researchers to under-load DNA in subsequent experiments, leading to failed reactions or inaccurate results.
RNA can also physically interfere with enzymatic reactions, such as the Polymerase Chain Reaction (PCR) or restriction enzyme digestion. In PCR, contaminating RNA may bind non-specifically to the DNA template or primers, reducing amplification efficiency. The quantity of RNA can also interfere by chelating necessary cofactors like magnesium ions, further inhibiting the activity of DNA polymerases.
Enzymatic Removal Using RNase
The most common and effective technique for removing RNA involves using Ribonuclease (RNase), an enzyme that specifically degrades RNA. RNase A, a widely used type, is an endoribonuclease that selectively cleaves phosphodiester bonds in single-stranded RNA following pyrimidine residues. This specific activity breaks down RNA into smaller fragments without causing damage to the double-stranded DNA template.
The procedure involves adding a DNase-free RNase A solution directly to the contaminated DNA sample, often at a final concentration around 10 micrograms per milliliter (µg/mL). The reaction is usually incubated near 37°C for 10 minutes to an hour, providing optimal conditions for RNase activity. This concentration ensures the complete degradation of the abundant RNA molecules.
Once the RNA is broken down, the RNase enzyme itself must be removed to prevent interference with downstream applications. Since RNase is a robust protein difficult to inactivate, simple heat treatment is often insufficient. Therefore, the sample is subjected to a secondary purification step, such as a phenol/chloroform extraction or a spin column cleanup. This successfully separates the large DNA from the small RNA fragments and the active enzyme.
Non-Enzymatic Purification Techniques
Non-enzymatic techniques are alternatives to enzymatic digestion, often integrated into modern DNA extraction workflows for convenience and speed. Commercial column-based purification kits are a prime example, relying on the differential binding of nucleic acids to a silica membrane. In high-salt binding buffers, DNA selectively binds to the column matrix, while smaller RNA molecules and other contaminants are washed away.
RNA fragments have a much lower molecular weight than genomic DNA strands and are too small to be efficiently retained by the silica filter under specified buffer conditions. The purified DNA is then eluted from the column using a low-salt buffer or water, resulting in a clean and concentrated sample. These spin-column methods are advantageous because they combine purification and RNA removal, eliminating the need for separate enzymatic digestion and cleanup.
Chemical precipitation methods can also be adjusted to favor DNA over RNA separation, though they are less common in routine laboratory work. When isolating plasmid DNA, using a higher salt concentration and a specific pH can differentially precipitate the larger plasmid DNA. This keeps the smaller, more soluble RNA fragments in the solution phase. This technique exploits the size and structural differences between the two nucleic acids chemically, rather than relying on an enzyme.
Assessing Sample Purity
After implementing an RNA removal strategy, confirming the purity of the final DNA sample is an essential quality control step. Spectrophotometric analysis is the standard method for initial assessment, utilizing the A260/A280 and A260/A230 ratios. Pure DNA is expected to have an A260/A280 ratio of approximately 1.8. A ratio significantly higher than this, closer to 2.0, strongly indicates residual RNA contamination, as pure RNA absorbs more strongly at 280 nm than pure DNA.
The A260/A230 ratio is also important, with ideal values falling between 2.0 and 2.2 for pure nucleic acid. A low A260/A230 ratio suggests contamination by residual organic compounds or salts used during extraction. The combination of both ratios provides a comprehensive chemical purity profile of the sample.
Visual confirmation can be achieved using agarose gel electrophoresis, which separates nucleic acids by size. A pure genomic DNA sample appears as a single, high-molecular-weight band near the top of the gel, indicating intact DNA. Contaminating RNA is much smaller and typically appears as a faint, fast-migrating smear near the bottom of the gel.