Now it's easy to make your RNA free of genomic DNA contamination and ready for RT-PCR.
DNA-free™ DNase Treatment & Removal Reagents contain RNase-free DNase, and an optimized DNase digestion buffer, to ensure safe, complete removal of contaminating DNA from any RNA sample. Also included is a unique DNase Removal Reagent which, after digestion, eliminates DNase in minutes — no more messy phenol extractions or heat inactivation procedures which can cause RNA loss or degradation.
DNA, contaminating RNA preparations, can serve as a template in PCR to produce a false positive signal from RT-PCR. Although false positives are easily identified by looking at the outcome of a "minus-RT" control, eliminating the DNA is not trivial. Here we discuss the problem of genomic DNA contamination in RT-PCR, the best methods to detect and remove it, and an innovative way to remove DNase I after DNase I treatment.
All Isolation Methods Result in Contaminating DNA
Figure 1. DNA Contamination in RNA Isolated by 5 Different Methods. Total RNA was isolated from mouse liver by the methods indicated. RNA (0.5 µg) underwent RT-PCR, or simply PCR (without reverse transcription), as indicated and aliquots of each reaction were electrophoresed on a 2% agarose gel and stained with ethidium bromide.
Lane RNA Isolation Method
- Single-reagent extraction method [e.g. TRIzol® Reagent (Invitrogen), TRI Reagent® (MRC), RNAzol® (Tel-Test), RNA Stat-60® (Tel-Test), RNAwiz™ (Ambion)]
- Glass fiber filter-binding method [e.g. RNeasy® (Qiagen), RNAqueous® (Ambion)]
- A multi-step guanidinium thiocyanate/acid phenol : chloroform extraction method (e.g. the Chomzynski and Sacchi procedure, Ambion's ToTALLY RNA™ Kit)
- Cetrifugation through a CsCl cushion
- Two rounds of oligo d(T) selection [e.g. FastTrack® RNA (Invitrogen), Poly(A)Pure™ Kit (Ambion)]
- Water control
Figure 1 shows that, regardless of the isolation method, gene specific product is synthesized in the absence of reverse transcriptase, indicating that none of these RNA isolation methods produce DNA-free RNA.
How Do You Know If Your RNA Is Contaminated with DNA?
PCR primers can be designed to control for genomic DNA contamination. Primers that span intron-exon boundaries amplify a product from contaminating DNA that includes the intron, making it much larger than the expected cDNA product. In fact, primers can be designed to span a genomic fragment large enough to make amplification from genomic DNA effectively impossible. Relying solely on primer design for the detection of DNA contamination, however, is not always enough. Pseudogenes may exist in your sample that can produce an amplified product of the same size as the expected cDNA product. (Pseudogenes arise from a processed mRNA that is reverse transcribed and then integrated into the genome; no introns are present). Figure 2 illustrates how primer design can be used to detect most DNA contamination, and why a "minus-RT" control remains necessary in any RT-PCR experiment.
Figure 2. Detection of DNA Contamination in RNA. Mouse liver RNA (0.5 µg) was used in RT-PCR. The S15 PCE primers span an intron-exon junction. Products from both the gene and the pseudogene for this message are detected in the minus-RT reaction.
Getting Rid of Contaminating DNA and the DNase Used to Destroy it
Commonly used methods for removal or inactivation of DNase after digestion include: heat inactivation, proteinase K treatment followed by phenol:chloroform extraction, chelation of essential ions with EDTA, and purification using a glass-filter binding method such as RNAqueous® (see the sidebar at right, "RNA Isolation for RT-PCR). Each of these inactivation or removal methods has its drawbacks.
Heat inactivation: Probably the most common method of DNase inactivation is heat treatment, typically for 5 minutes at 75°C. Although this method appears straightforward, the divalent cations in the DNase digestion buffer can cause (chemically-induced) strand scission of RNA when heated. Studies at Ambion have shown that much of an RNA sample is destroyed when heated to 80°C for 5 minutes in the presence of 2.5 mM MgCl2 and 0.1 mM CaCl2 (salts typically found in DNase I digestion buffer).
Proteinase K treatment and organic extraction: Proteinase K treatment followed by phenol:chloroform extraction is probably the most rigorous method for DNase inactivation and removal, but it is time-consuming, and organic extractions often cause some sample loss. Sample loss can be minimized by back extraction of the phenol:chloroform phase, but this adds another step to an already time-consuming procedure. Additionally, many people prefer to avoid working with hazardous phenol.
EDTA chelation of cations: The addition of EDTA to DNase digestion reactions chelates ions in the digestion buffer, that are required for DNase I activity. The DNase I can then be safely heat inactivated without loss of RNA. However, Mg2+ is needed for enzymatic activity of both the reverse transcriptase and the thermostable DNA polymerase. Thus the chelation capacity of the EDTA must be saturated with additional ions prior to subsequent enzymatic reactions. This can make the assembly of a simple reaction quite complicated.
RNA purification: Some filter-based RNA isolation methods treat with DNase directly on the filter after binding of the lysate. This treatment may not completely eliminate contaminating DNA because the DNase will not be in an optimal environment for digestion (traces of lysis solution and other contaminants may interfere with optimal digestion). Alternatively, RNA preparations that have been treated with DNase in solution, can be purified away from DNase over such columns. Although this technique adequately removes DNase from the prep, it requires both an extra step, and expensive materials.
RNA Isolation for RT-PCR
RT-PCR Experiments Using Total RNA isolated with the RNAqueous®-4PCR Kit. RNA was used as template in reverse transcription (RT) reactions or in mock RT reactions that did not contain reverse transcriptase. Ten percent of the resulting cDNA was amplified by PCR using S15 primers. No PCR product is seen from the minus-RT reactions, demonstrating the lack of DNA contamination in RNA isolated using Ambion's RNAqueous-4PCR Kit. The lanes to the right of the markers show the S15 RT-PCR product from the indicated samples.