Тhe quality of the RNA used for library generation is of the utmost importance. Put another way, high quality RNA will have the best chance of leaving the researcher with high quality data. There are many instances where researchers may find themselves in a place where high quality, high purity RNA starting material is impossible, such as working with FFPE samples. Thus, we can think of this through yet another lens: getting the most from what you have. Whether you are working with FFPE samples or cell lines, the goal is to get the most from what your starting material can offer. In many cases, DNase treatment is a key step in maximizing the quality of the RNA samples used for sequencing.
With few exceptions, RNA samples contain some amount of contaminating genomic DNA (gDNA). It is not surprising then, that many RNA extraction kits recommend, or even require DNase treatment of RNA samples before proceeding to challenging downstream applications, such as transcriptome analysis.
In RNA-Seq applications, random primers or short oligo(dT) primers cannot distinguish between RNA and DNA, and will also hybridize to residual gDNA. In addition, reverse transcriptases are promiscuous enzymes which are able to use DNA as template molecule. As a result, unwanted gDNA will be channeled through the entire RNA-Seq workflow, which can cause biases and quantification issues during the final data analysis steps. Therefore, it is critical to remove any residual gDNA to obtain the best quality data.
1. Genomic DNA Removal Methods
The most common means of DNA removal is by DNase digestion. DNase, short for Desoxyribonuclease, is a DNA-specific endonuclease that cleaves single- and double-stranded DNA, leaving behind 5’ phosphorylated oligonucleotide products. Because of this versatility it is used in a wide range of biological applications. It is important to note that DNase should be removed afterwards as residual DNase may also affect downstream reactions in the library preparation.
2. DNase Clean-up Methods
Remaining DNase should be removed from the sample before the experiment proceeds. If DNase is carried over into library preparation, primers initiating reverse transcription may be degraded ultimately affecting the efficiency of the library generation. The method of DNase clean-up is perhaps just as important for the sample quality as the use of DNase in the first place. To minimize DNase presence in the final RNA sample there are a number of options available, each with their own strengths and weaknesses.
3. Detecting gDNA in your Sample
When is DNase treatment required for your RNA sample and how can you decide if the gDNA really was efficiently removed? As you can imagine, due to the similarities between these nucleic acids, detecting gDNA in an RNA sample is difficult. But there are a few methods that can be used to determine whether or not gDNA is present:
➊ Optical Density (OD) measurements using a spectrophotometer and comparing emissions at different wave lengths can give you an indication of the purity of your RNA sample, e.g., an OD 260/280 value below 2 can indicate DNA contamination.
➋ Some spectrophotometric assays using fluorescent dyes specific for either RNA or DNA can distinguish between RNA and DNA within a sample.
➌ Agarose gel electrophoresis can also show gDNA in the high molecular weight region of the gel (Fig. 1A).
➍ Running the extracted RNA on a Fragment Analyzer using extended run time settings can reveal gDNA contamination in a similar way to that of the gel above: a high molecular weight “bump” in the trace indicates the presence of gDNA in the sample (Fig. 1B).
Figure 1 | Assessing your RNA sample for genomic DNA (gDNA). A) Stained agarose gel assessing RNA extracted using an RNA extraction method with (lane 1) and without gDNA removal (lane 2). A high molecular weight band is visible in lane 2, indicating the presence of genomic DNA. B) Fragment Analyzer trace with extended runtime assessing RNA extracted from white blood cells. A high molecular weight “bump” indicates gDNA contamination of the sample.
Most of these methods only detect rather high levels of gDNA presence in a sample. Unfortunately, RNA-Seq is so sensitive to gDNA contamination that the amount required to negatively impact the process falls below the detection threshold of some of the methods described above. This is a primary contributing factor to the popularity of DNase treatments in RNA-Seq workflows.
The best approach to detect residual gDNA and ensure the RNA preparation is indeed fully DNA-free is to run a PCR or qPCR with primer pairs for a specific set of marker genes. Commonly referred to as house-keeping genes, there are some popular genes such as GAPDH, genes encoding RNA-polymerase subunits, actin, rDNA loci, and others. As PCR can detect trace amounts of DNA from as low as one molecule, PCR is the method of choice to exclude gDNA contamination in workflows that require highly pure RNA that needs to be absolutely free of DNA.
4. To Treat or Not to Treat
The question remains, when is it necessary to treat the RNA preparation with DNase, and when is it unnecessary? In general, DNase treatment should always be considered for the following circumstances:
When can DNase treatment be omitted?
As you have seen, some RNA extraction procedures are more suitable to minimize gDNA carry-over than others, especially acidic phenol / chloroform extraction can remove gDNA from many standard sample types. Also, 3’ mRNA-Seq library preps tend to pick up less gDNA background than random primed whole transcriptome library preps, due to the fact that 3’-Seq methods rely on poly(A) stretches for priming. When both are used in combination and the RNA is of high quality and derived from an unproblematic sample type, the risk of compromising your RNA-Seq experiment with DNA contamination is minimal.
Those running targeted sequencing experiments can breathe a sigh of relief, as it were. DNase treatment is not needed for most RNA-Seq applications using targeted primers for specific genes of interest. The risk of contamination with accessible gDNA for exactly this location is low and genomic loci for primer binding are often far from the respective regions of the transcript (e.g., due to the presence of introns), and thus DNase treatment is not required.