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
There is no RNA isolation method that consistently produces RNA free from genomic DNA without the use of DNase. To illustrate this, RT-PCR was performed on mouse liver RNA isolated by several different methods:
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 g) 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 RN A Isolation Method 1 single-reagent extraction method [e.g. TRIzol Reagent (Life Technologies), TRI Reagent (MRC), RNAzol (Tel-Test), RNA Stat-60 (Tel-Test), RNAwiz (Ambion)] 2 glass fiber filter-binding method [e.g. RNeasy (Qiagen), RNAqueous (Ambion)] 3 a multi-step guanidinium thiocyanate/acid phenol:chloroform extraction method (e.g. the Chomzynski and Sacchi procedure, Ambion's ToTALLY RNA Kit) 4 centrifugation through a CsCl cushion 5 two rounds of oligo d(T) selection [e.g. FastTrack RNA (Invitrogen), Poly(A)Pure Kit (Ambion)] 6 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?
How can you test for DNA contamination in RNA samples? The best way is to include a "minus-RT" control for each RNA sample in an RT-PCR experiment. If a PCR product is generated from an RNA sample that was not reverse transcribed (minus-RT control), then the product was amplified from contaminating DNA. Contaminating DNA may come from either the RNA preparation or from the RT-PCR reagents. A minus-template control for the PCR distinguishes between these possibilities.
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 g) 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
Because virtually all RNA samples have trace amounts of contaminating DNA, most protocols specify DNase treatment for RT-PCR applications. DNase I treatment is clearly the best way to rid an RNA sample of contaminating DNA. However, some preparations of DNase may be contaminated with RNases, and the DNase must be completely inactivated prior to RT-PCR so that it doesn't degrade newly synthesized DNA. Unfortunately, removal or inactivation of this enzyme is problematic; DNase removal methods can be inconvenient, ineffective and even detrimental to RNA integrity.
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 75C. 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 80C 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
With the RNAqueous-4PCR Kit, you can isolate RNA free of genomic DNA contamination from samples as small as 100 cells or 1 mg of tissue. The kit contains reagents for the phenol-free isolation of RNA, and reagents to remove contaminating DNA. The kit also contains plastic pestles designed for disruption of small tissue samples, using microfuge tubes as mortars. The RNAqueous procedure involves disrupting tissues or cells in a guanidinium-based lysis solution, binding the RNA to a glass fiber filter, washing the filter to remove contaminants, and recovering the RNA in a small volume of elution solution. Finally, the RNA is treated with the DNase treatment and removal reagents included in the kit yielding RT-PCR ready RNA.
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.
DNA-free Removes the DNA, and Then Removes the DNase
DNA-free DNase Treatment & Removal Reagents are one of Ambion's latest tools designed to simplify RNA preparation for RT-PCR. With DNA-free, genomic DNA contamination can be removed from any RNA preparation without incurring RNA loss or risk of degradation. The DNA-free DNase Treatment & Removal Reagents provide RNase-free DNase I and optimized DNase Reaction Buffer for the complete digestion of contaminating DNA in RNA samples. Ambion's DNase I is prepared with ultra pure reagents and is not released for sale until it is shown to be both highly effective at eliminating DNA, and devoid of RNase activity. The 10X DNase Reaction Buffer is designed for optimal DN ase I activity. DNA-free also includes a novel DNase Removal Reagent to quickly eliminate the DNase after treatment. This unique reagent effectively removes all traces of DNase and divalent cations from the reaction mixture after DNA digestion is complete. The DNase/cation removal step is fast taking only three minutes to complete. After DNase digestion, The Removal Reagent is added, the tube flicked to mix, and the solution incubated for 2 minutes at room temperature. DNase and ions are bound by the DNase Removal Reagent which is spun out with a quick centrifugation leaving the RNA in the supernatant ready for RT-PCR. This simple method avoids messy organic extraction or heat inactivation of DNase I that may put your RNA at risk. DNA-free DNase Treatment & Removal Reagents are also available as components of the RNAqueous-4PCR Kit (see sidebar, "RNA Isolation for RT-PCR").