Make Your RNA Free of Genomic DNA Contamination and Ready for RT-PCR

Invitrogen 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.

DNA-free™ DNase Treatment & Removal Reagent包括无RNase的DNase以及优化的DNase消化缓冲液，能确保安全、完全地去除RNA样品中的污染DNA。该试剂还包括一种独特的DNase Removal Reagent ，可在消化后数分钟内去除DNase，因此无需进行操作繁杂且容易造成RNA损失或降解的苯酚萃取或热灭活等步骤。

RNA制备过程中的污染DNA可在PCR过程中作为模版，从而使RT-PCR得到假阳性结果。尽管通过观察无逆转录对照结果可以很容易地判定假阳性结果，但去除污染DNA仍然是非常重要的。本文我们将讨论RT-PCR过程中基因组DNA污染问题、检测和去除DNA污染的最佳方法，以及在进行DNase I处理后去除DNase I的创新方法。

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 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

1. Single-reagent extraction method [e.g. Invitrogen TRIzol Reagent, Invitrogen TRI Reagent Solution, RNAzol® (Tel-Test), RNA Stat-60® (Tel-Test), Invitrogen RNAwiz]
2. Glass fiber filter-binding method [e.g. RNeasy® (Qiagen), Invitrogen RNAqueous]
3. A multi-step guanidinium thiocyanate/acid phenol : chloroform extraction method (e.g. the Chomzynski and Sacchi procedure, Invitrogen ToTALLY RNA Kit)
4. Centrifugation through a CsCl cushion
5. Two rounds of oligo d(T) selection [e.g. Invitrogen FastTrack RNA, Invitrogen Poly(A)Pure Kit]
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.

在不使用DNase的情况下，所有RNA提取方法都无法保证得到无基因组DNA污染的RNA。为了说明这一点，我们对利用了不同提取方法得到的小鼠肝脏RNA进行了RT-PCR。

图 1.使用5种不同方法提取得到的RNA中的DNA污染情况。我们使用如图所示的不同方法提取小鼠肝脏中的总RNA，然后对0.5ug RNA进行如图所示的RT-PCR或PCR（无逆转录），之后各取一份反应液在2%的琼脂糖凝胶上进行电泳并进行溴化乙锭染色。

RNA提取方法

1. 单一试剂提取法（如TRIzol Reagent (Invitrogen)、TRI Reagent (MRC)、RNAzol (Tel-Test)、RNA Stat-60 (Tel-Test)、RNAwiz™ (Ambion)）
2. 玻璃纤维滤膜结合法（如RNeasy (Qiagen)、RNAqueous (Ambion)）
3. 多步骤的硫氰酸胍/酸酚氯仿提取法（如Chomzynski 和 Sacchi 步骤和Ambion's ToTALLY RNA™ 试剂）。
4. CsCl密度梯度离心法
5. 两轮oligo d(T)筛选法（如FastTrack RNA (Invitrogen)和Poly(A)Pure™ Kit (Ambion)）
6. 水对照

从图1可以看出，不管是哪种提取方法，在无逆转录酶的情况下都会有基因特异性产物的合成，这意味着这些RNA提取方法都不能得到无DNA污染的RNA。

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 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.

如何检测RNA样品中是否有DNA污染？最好的办法是在RT-PCR实验中为每个RNA样品提供无逆转录对照。如果PCR产物来自于未经逆转录的RNA样品（无逆转录对照），则该产物即是由污染DNA的扩增而来。污染DNA既可能来源于RNA制备过程，也可能来源于RT-PCR中所用的试剂。设置无模板的PCR对照可以很好地区分这2种情况。

可以设计PCR引物来作为基因组DNA污染分析的对照。可扩增内含子-外显子区域的引物能够扩增含有内含子的污染DNA的产物，且比预期的cDNA产物更大。实际上，可以优化引物设计，使其几乎不能扩增基因组片段。然而，单纯依赖引物设计来检测DNA污染有时是不够的。若样品中含有假基因，则假基因也可以得到与预期cDNA产物相同大小的扩增产物（例如，假基因可能来自经过剪切等修饰的mRNA，在反转录后整合回基因组中，因此不含内含子）。图2描述了如何设计引物来检测大多数DNA污染，以及为什么在所有RT-PCR实验中仍然需要无逆转录对照。

图 2. 检测RNA中的DNA污染。 使用小鼠肝脏RNA (0.5 µg) 作为RT-PCR模板，S15 PCE引物可以扩增出内含子-外显子产物。在无逆转录对照反应中检测到了来自基因和假基因的产物。

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 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 here 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.

几乎所有的RNA样品都有DNA污染，因此大多数实验方案在RT-PCR实验中会用到DNase。DNase I显然是去除RNA样品中DNA污染的最好办法。然而，有些DNase在制备过程中会被RNase污染。另外，在RT-PCR之前必须使DNase完全失活，以免降解新合成的DNA。不幸的是，去除或失活这种酶会带来很多问题，DNase去除方法操作起来不方便、效果不佳，甚至会损伤到RNA的完整性。

常用的去除或失活消化处理后的DNase的方法包括：热灭活、蛋白酶K处理后进行酚氯仿提取、EDTA螯合其发挥作用所必须的阳离子以及利用玻璃纤维滤膜结合法纯化（如RNAqueous，参见下文中的“用于RT-PCR的RNA提取”）。但是这些失活或去除方法都有各自的缺点。

热灭活: 热灭活可能是最常用的失活DNase的方法，只需在75°C处理5分钟。尽管这种方法直接简单，但加热时DNase消化缓冲液中的二价阳离子会引起化学诱导的RNA链剪切。Ambion的研究发现，在含有2.5 mM MgCl2和0.1 mM CaCl2（DNase I消化缓冲液中经典的盐离子组分）的条件下，在80°C加热5分钟后，多数RNA样品都会被破坏。

蛋白酶K处理和有机提取: 先进行蛋白酶K处理然后进行酚氯仿提取可能是最严格的去除和失活DNase的方法，但这种方法非常耗时，且有机提取通常会造成样品损失。虽然之后可以通过酚氯仿相的再提取来最大限度减少样品损失，但对于本来就耗时的流程而言这又新添了一个步骤。此外，许多研究人员会尽量避免操作有害的酚试剂。

EDTA螯合阳离子: 在DNase消化反应液中加入EDTA，可以螯合DNase I保持活性所必需的离子，这样可以很快使DNase I安全失活，并且不会造成RNA损失。然而，逆转录酶和热稳定的DNA聚合酶都需要Mg2+来保持活性，因此在酶促反应前必须加入额外的离子使EDTA的螯合能力达到饱和，再进行后续的酶促反应。而这会使本来简单的反应变得复杂。

RNA纯化: 一些使用滤膜的RNA提取法会在滤膜与裂解液结合后直接处理DNase。这种处理方法可能无法完全去除污染DNA，因为DNase不会处在最优的消化环境中（痕量的裂解液及其他污染物可能会干扰最优的消化环境）。也可对使用DNase处理的RNA制备溶液进行纯化，分离出其中的DNase。然而这种技术虽然能充分去除DNase，但需要额外的步骤以及昂贵的材料。

With the Invitrogen™ 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 the RNAqueous-4PCR Kit. The lanes to the right of the markers show the S15 RT-PCR product from the indicated samples.

Learn more about RNA isolation for qRT-PCR

使用RNAqueous-4PCR Kit，可以从小至100个细胞或1mg组织的样品中提取得到无基因组DNA污染的RNA。该试剂盒包含用于无酚提取RNA的试剂以及去除污染DNA的试剂，另外还包括研磨小组织样品的塑料棒，能配合离心管进行研磨。RNAqueous的提取步骤包括在胍盐裂解液中破碎组织或细胞、结合RNA至玻璃纤维滤膜上、清洗滤膜上的污染物、加入少量洗脱液回收RNA以及使用试剂盒中的DNase处理和去除试剂处理，最终得到可用于RT-PCR的RNA。

使用由RNAqueous-4PCR Kit提取得到的总RNA进行RT-PCR实验。以RNA为模板，分别进行RT反应及不含逆转录酶的假RT反应。使用S15引物对以上反应中得到的十分之一的cDNA进行扩增。在无逆转录反应中没有发现PCR产物，表明使用Ambion的RNAqueous-4PCR Kit提取到的RNA中没有DNA污染。分子量标准品右边的电泳区带显示了来自样品的S15 RT-PCR产物。

DNA-free DNase Treatment & Removal Reagents are one of our 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 DNase 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").

DNA-free DNase Treatment & Removal Reagent是Ambion最新推出的用于简化用于RT-PCR的RNA的制备的工具，可以去除任何RNA中的基因组DNA污染，并且不会造成RNA损失或降解。该试剂包含RNase-free DNase I以及优化的DNase Reaction Buffer，可完全消化RNA样品的中的污染DNA。Ambion的DNase I使用超纯试剂制备，我们在确认其能非常有效地去除DNA及失活RNase之后才将其推出上市。10X DNase Reaction Buffer设计用于发挥DNase I的最佳活性。DNA-free还包含一种创新的DNase Removal Reagent，可以在DNase处理完成后快速去除DNase。该试剂在DNA消化完成后，可有效去除反应液中所有的痕量DNase和二价阳离子。DNase/离子去除步骤非常快，仅三分钟就可完成。DNase消化完成后，加入Removal Reagent并摇匀，然后在室温下孵育2分钟。待DNase Removal Reagent与DNase和离子结合后，对DNase Removal Reagent进行快速离心，使RNA保留在上清用于后续的RT-PCR。该简易方法无需进行麻烦的有机提取或对DNase I热灭活，不会影响到RNA本身。DNA-free DNase Treatment & Removal Reagent也包含在RNAqueous-4PCR Kit中提供（参见上文“用于RT-PCR的RNA提取”）。