Purification and quantification of DNA and RNA is essential for key downstream applications in life sciences, clinical diagnostics, forensics and drug discovery research.
Many different kits are available for the extraction and purification of DNA and RNA. These have varying components depending on the starting material and the application of the purified product. The purified DNA and RNA can be quantified using electrophoresis or spectrophotometers.
Purification and quantification of DNA and RNA are essential for key downstream applications in life sciences, clinical diagnostics, forensics and drug discovery research.
Many different kits are available for the extraction and purification of DNA and RNA. These have varying components depending on the starting material and the application of the purified product. The purified DNA and RNA can be quantified using electrophoresis or spectrophotometers.
This SelectScience® how-to-buy eBook provides you with an overview of the key technologies and considerations when purchasing kits for DNA and RNA purification and analysis.
1.1 Sample Source
The starting material for DNA extraction often determines the types of buffers and mechanical steps you need to use. The sources of DNA vary by field types. For life sciences, it can include bacteria, cells, rodent tails, human tissues, formalin-fixed human tissues; for clinical laboratories it can include blood/plasma samples, stools, biopsies and fine needle aspirates, while for forensics, it can include dried blood spots, buccal swabs, hair and fingerprints.
Below, we’ve listed some key considerations for different sample sources.
(i) Tissue Samples
Isolation of DNA/RNA from tissue samples such as heart, skeletal muscle, skin or tail tissue can be difficult due to the abundance of contractile proteins, connective tissue, and collagen. In order to remove these proteins, which can interfere with DNA/RNA isolation, the sample needs to be treated with a protease or phenol-containing lysis reagent.
(ii) FFPE Tissue Samples
Formalin-fixed paraffin-embedded tissues represent a valuable and extensive source of material for genetic and molecular analyses across a wide range of cohorts. With an increasing number of researchers turning toward molecular analysis of FFPE samples, it is becoming increasingly important to develop specific protocols that take into consideration the unique nature of these samples.
Due to fixation and embedding conditions, nucleic acids in FFPE samples are usually heavily fragmented and chemically modified by formaldehyde. The degree of fragmentation depends on the type and age of the sample and on the conditions for fixation, embedding and storage of the sample. Although formaldehyde modification cannot be detected in standard quality control assays, such as gel electrophoresis or lab-on-a-chip analysis, it does strongly interfere with enzymatic analyses. Steps can be taken to minimize the effects of FFPE storage on the nucleic acids, such as use of thin tissue samples, not over-fixing tissue slices, use of quality paraffin embedding reagents, avoidance of sample staining, and storage at low temperatures.
(iii) Blood Samples
Nucleic acid isolation from blood requires a method to provide high-quality DNA or RNA without contaminants or enzyme inhibitors. Blood contains a number of enzyme inhibitors that can interfere with downstream analysis. In addition, common anticoagulants used in the collection step, such as heparin and EDTA, can interfere with downstream assays.
Erythrocytes, i.e. red blood cells (RBCs), do not contain nuclei and are therefore not important for DNA/RNA. The target of isolation from whole blood is leukocytes, i.e. white blood cells (WBCs). Since healthy blood contains approximately 1,000 times more RBCs than WBCs, removing the RBCs simplifies the isolation process. This can be accomplished by selective lysis of RBCs, which are more susceptible to hypotonic shock than WBCs and burst rapidly in the presence of a hypotonic buffer.
A common alternative to RBC lysis is Ficoll density-gradient centrifugation. In contrast to RBC lysis procedures, Ficoll density-gradient centrifugation only recovers mononuclear cells, i.e. lymphocytes and monocytes, and removes granulocytes. Mononuclear cells isolated by Ficoll density-gradient centrifugation can then be processed for nucleic acid isolation.
(iv) Plant Samples
Isolation of DNA and RNA from plant material presents a special challenge: several plant metabolites have chemical properties that are similar to nucleic acids, making them difficult to remove from sample preparations. Therefore, commonly used techniques require adaptation before they can be used with plant samples. Co-purified metabolites, such as polysaccharides, polyphenolics, and flavones, and contaminants introduced by the purification procedure, such as salts or phenol, can inhibit enzymatic reactions or cause variations in UV spectrophotometric measurements and gel migration. Another consideration in the process of nucleic acid isolation is potential pipetting errors due to increased viscosity.
(v) Viral Samples
(v) Viral Samples Viruses from clinical samples are often isolated from cell-free body fluids, where their titer can be very low. Virus particles may need to be concentrated before nucleic acid isolation by ultracentrifugation, ultrafiltration, or precipitation.
1.2 Downstream Applications
When selecting an extraction kit for nucleic acids, it’s important to note that the resulting quality and purity of the extracted nucleic acids should be suitable for your intended downstream application. Different applications, ranging from routine PCR genotyping, to next-generation sequencing (NGS), to restriction endonuclease digestions are all influenced by the purity of the nucleic acid yield.
1.3 Sample Quantity
Your choice of nucleic acid extraction kit will also depend on the quantity of the starting sample. For example, confluency of cultured cells, milligrams of tissue biopsies, or volume of blood, and so on. For precious samples, ensure that the kit is first tried on control samples and optimized for use before proceeding.
In order to extract the DNA from a cell or tissue sample, the cells need to be disrupted by cell lysis. This is usually achieved by chemical and physical methods, such as homogenization or sonication. During the cell lysis process, the membrane lipids are removed by adding a detergent or surfactants. A protease is often used to remove cellular or histone proteins bound to the DNA, and RNases are often used to remove unwanted RNA. The raw DNA product is then purified to remove the detergents, proteins, salts and reagents used during cell lysis step.
2.1 Common Procedures
The most commonly used procedures are:
1. Ethanol precipitation, usually by ice-cold ethanol or isopropanol. Since DNA is insoluble in these alcohols, it will aggregate together, forming a pellet upon centrifugation. Precipitation of DNA is improved by increasing ionic strength, usually by adding sodium acetate.
2. Phenol-chloroform extraction, in which phenol denatures proteins in the sample. After centrifugation of the sample, denatured proteins stay in organic phase. The aqueous phase containing nucleic acid is then mixed with the chloroform which removes phenol residues from solution.
3. Mini-column purification relies on the fact that the nucleic acids are adsorbed to a solid phase such as silica, depending on the pH and the salt content of the buffer.
There are refinements to this technique, which include adding a chelating agent to sequester divalent cations such as Mg2+ and Ca2+; this prevents enzymes such as DNase from degrading the DNA.
After isolation, the DNA is usually dissolved in slightly alkaline buffer or in ultrapure water.
|DNA extraction technologies||Anion-exchange||Silica membrane||Magnetic particles|
|Technology||Solid-phase, anion-exchange chromatography||Selective adsorption to silica membranes||Binding to magnetic silica particles under controlled ionic conditions|
|Binding/Elution||Binding and elution: variable salt and pH Requires alcohol precipitation||Binding: high salt Elution: low salt Ready-to-use eluate||Binding: high salt Elution: low salt Ready-to-use eluate|
|Application||Delivers ultrapure, transfection-grade DNA for optimal results in sensitive applications||Delivers high-purity nucleic acids for use in most downstream applications||Delivers high-purity nucleic acids for use in most downstream applications|
The most commonly used DNA extraction kits have a variation of three different types of technology, as outlined in Table 1.
2.2 Common DNA Extraction Technologies:
Anion-exchange methods yield DNA of a purity and biological activity equivalent to at least two rounds of purification in CsCl gradients, in a fraction of the time. Purified nucleic acids are of the highest possible quality and are ideal for sensitive downstream biological applications, such as transfection, microinjection, sequencing, and gene therapy research.
Silica-membrane technology yields high-purity nucleic acids, suitable for most molecular biology and clinical research applications, such as restriction digestion, ligation, labeling, amplification, and radioactive and fluorescent sequencing.
Magnetic-particle technology also yields high-purity nucleic acids suitable for most molecular biology applications. Magnetic-particle technology can often be automated to enable fast and economical nucleic acid purification procedures. Magnetic beads are rapidly being adopted in genomics and are replacing filtration kits. Corning® offers a comprehensive line of magnetic bead-based kits for fast, rapid isolation of genomic DNA from various starting samples.
2.3 DNA Isolation Considerations:
(i) Plasmid DNA Isolation
Plasmid DNA isolation is a basic technique that is performed in most molecular biology laboratories. Multi-sample processing is often required to complete both plasmid isolation and subsequent downstream experimentation.
The Genopure Plasmid Maxi Kit by MilliporeSigma helps prepare transfection-grade plasmid DNA in large quantities (up to 500 μg plasmid) from bacterial cultures. The isolated plasmid is suitable for most molecular biology applications such as transfection, Southern blotting, sequencing, PCR, restriction digestion and cloning.
This downloadable application note describes how to obtain high-yield plasmid DNA using miniprep. The GenElute™ Five-Minute Plasmid Miniprep by MilliporeSigma offers a quick, streamlined protocol to yield up to 5µg of high-quality DNA in five minutes.
(ii) Genomic DNA Isolation
Genomic DNA can be extracted from a variety of samples including tissue, cells, blood, serum, plants and forensic samples. Kits compatible with high-throughput capabilities are available for your automation needs.
(iii) Plant DNA Isolation
There are a number of suitable kits for extraction of DNA from plant matter. Again, consider the sample and the purity of the extracted DNA required for the analysis and downstream application. QIAGEN's DNeasy 96 Plant Kit yields up to 15µg per well of total cellular DNA from plant tissue.
Purified RNA is used for many downstream applications. DNA extraction methods cannot be directly applied to RNA, as RNA is structurally very different from DNA. RNA is single-stranded, while DNA is mostly double-stranded. It is often difficult to isolate intact RNA. RNases, a group of enzymes that degrade RNA molecules, are abundant in the environment, including on hands and on surfaces, and it is difficult to completely remove/destroy RNases. RNA isolation therefore requires cautious handling of samples and good aseptic techniques. It is important to use only RNase-free solutions during the extraction, as well as RNase-free pipette tips and glassware.
Ribonucleases (RNases) are very stable and active enzymes that generally do not require cofactors to function. Since RNases are difficult to inactivate, and even minute amounts are sufficient to destroy RNA, no plastic or glassware should be used without first eliminating possible RNase contamination. Great care should be taken to avoid inadvertently introducing RNases into the RNA sample during or after the purification procedure. In order to create and maintain an RNase-free environment, precautions must be taken during pre-treatment and use of disposable and non-disposable vessels and solutions while working with RNA.
Isolation of noncoding RNAs, functional RNA molecules that do not translate into proteins, may be desired in some applications. Such RNAs include tRNA and rRNA, as well as small nucleolar RNAs (snoRNA), microRNAs (miRNA), short interfering RNAs (siRNA) and piwi-interacting RNAs (piRNA). They are often involved in the regulation of gene expression. Specialized kits for isolation of noncoding RNAs are available, such as the High Pure miRNA Isolation Kit by MilliporeSigma.
In a recent SelectScience® interview, Dr. Michael Bianchi, of Empire Genomics, shares his experience at isolating RNA from FFPE tissue samples.
RNA isolation from bacteria: Bacterial mRNAs differ from eukaryotic mRNAs in a number of essential features. Prokaryotic mRNAs have no 5' cap and only rarely have poly-A tails. The absence of a poly-A tail means that mRNA isolation by hybrid capture is not possible. In addition, oligo-dT primers cannot be used to prime first-strand cDNA synthesis, so random primers need to be used instead. In addition, bacterial mRNAs are highly unstable, with an average half-life of about three minutes for fast-growing bacteria. Sometimes the bacterial mRNA begins to degrade while it is still being translated. This can be a big problem for researchers trying to isolate mRNA from bacteria. Since mRNAs are very rapidly turned over in bacteria, gene expression studies are even more difficult in prokaryotes than in eukaryotes. To accurately preserve gene expression patterns and to maximize the amount of fully intact mRNA isolated, samples need to be stabilized prior to sample harvesting and processing.
Cell-free RNA in plasma, serum or other bodily fluids: RNA, especially miRNA, associated with proteolipids (vesicles) or proteins can be found in bodily fluids, including plasma, serum, urine and cell culture supernatants. The concentration is much lower than that of cellular RNA, but approximately tenfold higher than cell-free DNA in human plasma. The RNA is relatively stable, with a half-life of about two days in human whole blood. Nonetheless, the RNA can be degraded by repeated freeze-thaw cycles. As with viral RNA in cell-free body fluids, addition of carrier RNA may be necessary during RNA isolation of this RNA.
4.1 Gel Electrophoresis
Gel imaging and nucleic acid binding dyes are widely used in today’s life science laboratories to visualize DNA fragments and identify mutations using agarose gel electrophoresis. Employed routinely for genotyping and animal transgenics, the nucleic acid staining dyes currently available can detect as little as 20 picograms of dsDNA or 3 nanograms of RNA.
This application note by UVP describes a method for safer visualization of DNA fragments, protecting both researchers and their DNA samples from damaging UV rays. Stay up-to-date with the latest in electrophoresis by visiting our life sciences community on SelectScience.
Quantification of nucleic acid samples prior to analysis is an important step in order to verify suitability for downstream molecular applications, such as qPCR, RT-qPCR, SNP genotyping and DNA sequencing.
As you make a decision on what spectrophotometer works best for your laboratory, here are a few considerations:
Cuvette-based versus microvolume spectrophotometer: Increasingly, researchers are moving towards microvolume spectrophotometers so as to use limited volume of their precious samples when quantifying nucleic acids. In a multi-functioning lab, it is possible that cuvette-based absorbance measurements are also often made, especially for protein-based applications. Depending on your projects, you can either consider a microvolume-only spectrophotometer or a dual mode one, where users can toggle between using a cuvette or making microvolume measurements, as needed.
Concentration versus purity measurements: Nucleic acid samples display a characteristic absorption spectrum at 260 nm. This yields the concentration of the sample. The purity of your sample is determined by the ratio of the absorbance at 260 nm and 280 nm (A260/A280), where the absorbance at 280 nm detects proteins present from the nucleic acid extraction process. A secondary measurement of contamination is the ratio of A260/A230, where the absorbance at 230 reflects guanidine and phenol residues present from the extraction process.
Built-in apps on spectrophotometers can not only provide the precise concentration without the need for dilutions, but also give feedback on the purity of the sample. With the Sample ControlTM technology, for example, if the sample is not clean, a warning symbol will be displayed providing information about potential contaminants to help optimize the purification steps in the extraction protocol.
|Nucleic Acid Type||Approximate A260/A280 ratios|
Novel spectrophotometers can offer concentration as well as purity measurements in the same readouts, so you get the most information for your downstream application choices. The NanoPhotometer® NP80, for example, offers a pre-programmed ‘nucleic acid’ method with options to measure all types of nucleic acids like dsDNA, ssDNA, RNA, miRNA and oligos, all with a single measurement.
Mobility: Traditionally, spectrophotometers have remained anchored in one corner of the lab, while you need to carry your sample tubes, pipettes, pipette tips and wipes to a fixed location. If, however, your lab uses particular samples involving a high risk of contamination, such as isolated RNA, it might be suitable to consider a portable, mobile spectrophotometer that can be brought to your DNase/RNase-free benchtop as and when you desire. These would also help busy facilities such as genotyping centers or DNA cores at universities. Portable spectrophotometers work on rechargeable battery packs. In the off-chance that your facility has a power cut, these enable your experiments to keep going.
Data storage and sharing: In a large team or in a shared facility, data storage often becomes a key consideration. In shared rooms, where all the data is stored on a single drive, it often becomes difficult to access your data if the instrument is being used by another user. Enquire about options for data storage and sharing before purchasing. Modern day spectrophotometers offer data transfer over Wi-Fi, ethernet or USB connections, so you can easily access your own readouts any time. An interface option such as a REST API enables you to transfer data, auto-populate fields and control the spectrophotometer from your laboratory information management system (LIMS). Comprehensive data storage and sharing packages are offered by NanoPhotometer®. Autosave, network functionality and other user-friendly features will help streamline your workflow, saving crucial time in the lab.
Recalibration and servicing: Spectrophotometers can be sold with service packages. Although these packages tend to be expensive, they are often necessary to cover the cost of recalibration and reconditioning of the instrument. Spectrophotometers utilizing a stepper motor to move between pathlengths will experience pathlength drift over time. To maintain accuracy, these units will require recalibration. Most manufacturers recommend a recalibration once a year. Consider these future maintenance issues before investing in a spectrophotometer.
If you notice a spectrophotometer is listed as ‘recalibration free’, you can request a letter from the manufacturer stating that fact. Such a letter would come in handy should there be a reason to recalibrate the spectrophotometer in the future.
Some spectrophotometers have now been designed to completely eliminate the need for recalibration. For example, the NanoPhotometer® NP80 offers two precisely defined pathlengths using fixed anchor points that do not change over the lifetime of the instrument. A magnet is used to move between the two pathlengths, eliminating the need for a motor function. Such a magnetic system ensures accuracy and prevents internal mechanical strain, thereby increasing the longevity of the instrument and making it recalibration-free for its lifetime..
Figure 1: The NanoPhotometer® NP80 uses fixed anchor points to offer two pathlengths. The magnetic system ensures accuracy and prevents internal mechanical strain, thereby eliminating the need for recalibration over the lifetime of the spectrophotometer.
Request a demo: With the advent of touch screens, cloud storage and smaller footprints, spectrophotometers too have become modern and innovative. Did you know that some spectrophotometers are now compatible with your cell phones and tablets across different operating systems? Or that the touch screens, as with the NP80, are optimized to work with your lab gloves on? A lot of technological development has happened since the traditional cuvette-based systems were launched decades ago. You can request a demo with the manufacturers of the latest spectrophotometers and test them for your lab before you make an investment solely based on traditional wisdom.
SelectScience® has plenty of resources you can use when deciding the best spectrophotometer choice for your lab.
In this video, see what scientists from around the world have to say about using Implen’s NanoPhotometer for their unique research applications and laboratory needs.
We interviewed Dr. Stephen Chanock, the Director of the National Cancer Institute (NCI) of the NIH, on how his lab performs retroactive studies over several decades using FFPE samples. Read the interview here.
Dr. Michael Bianchi, Empire Genomics, takes us back to basics: the foundation to complex experiments begins with simple, reproducible nucleic acid extraction methods. Read the interview here.
Read an interview with Dr. Laura Lindsey-Boltz, Associate Research Professor in the Nobel Prize-winning Sancar laboratory, and find out about their research on DNA damage and repair.
Plus, SelectScience® has numerous product reviews shared by your peers.
In this SelectScience® interview, Dr. Voula Kodoyianni, product manager at Thermo Fisher Scientific, describes the unique features of the NanoDrop One and how it can help advance your research.
All your FAQs on FFPE tissues are answered: Read this helpful Q&A with Dr. Jung Doh, application scientist at Beckman Coulter Life Sciences.
High risk, high pay-off: This video profiling Dr. Alejandra Perotti, University of Reading, discusses the sensitivity required to quantify DNA extracted from a single mite, especially when the samples belong to a collection started by Charles Darwin.
Extracted and purified DNA is used for many downstream applications in a number of fields, including clinical diagnostics, basic research, life sciences and forensics.
Depending on the purity and quantity of the nucleic acids, applications include PCR and RT-PCR, restriction digestion and cloning, sequencing, array CGH, gene expression microarray, SDS-PAGE, Northern and Southern blotting. Some of these are covered here.
PCR & RT-PCR
The polymerase chain reaction (PCR) is a technique used to amplify DNA sequences, and is widely used for many applications, such as molecular biology, microbiology, genetics, diagnostics, clinical laboratories, forensic science, environmental science, food science, hereditary studies and paternity testing. The downloadable PCR Technology Buying Guide provides more information about PCR kits and thermal cycling equipment.
Northern Blot Analysis
The Northern blot is a technique used in molecular biology research to study gene expression by detection of RNA (or isolated mRNA) in a sample. With Northern blotting it is possible to determine gene expression levels during differentiation, morphogenesis, as well as abnormal or diseased conditions. This application note compares different membranes and fixation methods for Northern blot analyses.
RNA sequencing, also called whole transcriptome shotgun sequencing (WTSS), is a technology that uses the capabilities of next-generation sequencing to reveal a snapshot of RNA presence and quantity from a genome at a given moment in time. This note outlines the various applications for high-end mRNA sequencing, and here is an overview of single-cell RNA-seq solutions from Illumina.
Microarray-based gene expression hybridization is a powerful and proven technique for studying differential gene expression signatures, and is especially valid for cancer research. This method describes how the sciPOLY3D by Scienion enables covalent and robust immobilization of unmodified DNA oligonucleotides for multiplex detection of RNA and DNA analytes.
Next-generation sequencing is a necessary requirement for today’s high-throughput research and clinical needs. One of the bottlenecks for NGS is the amount of time and resources required for library preparation; this is true whichever sequencing instrument you choose. For a comprehensive review of NGS sequencers and library construction kits, please read the SelectScience Next-Generation Sequencing How-to-Buy eBook.
DNA and RNA extracted from various materials, plants, hair, cells and tissues from a crime scene are used to gain data and evidence about the crime. Technologies for PCR, sequencing, electrophoresis and other analysis tools are used frequently.
For example, in 2015, the FBI granted approval for the Promega PowerPlex Fusion 6C System for use in laboratories that generate DNA records for the National DNA Index System (NDIS). A noteworthy mention from SelectScience editorial archives is the cutting-edge DNA coding technology used by leading forensic geneticist, Dr. Jari Louhelainen, of Liverpool John Moores University, UK, to discover the identity of the notorious London serial killer ‘Jack the Ripper’, almost 126 years after the last murder victim was discovered. Plus, learn how 50-year old DNA samples are being analyzed to identify the missing from the Vietnamese war.
DNA/RNA can be extracted from a wide range of biological patient specimens such as blood, plasma, swabs, saliva, urine, stool and biopsy preparations. The first application of PCR in clinical diagnostics was in testing for genetic disease mutations. Molecular diagnostic laboratories routinely test for a large variety of mutations, such as those found in hematological disorders, for example Factor V Leiden, cancer mutations such as epidermal growth factor receptor gene (EGFR) in non-small cell lung cancers, KRAS in colorectal cancer, and prenatal testing by amniocentesis, to name a few. Extracted genetic material can also be used for procedures such as tissue typing in transplant patients.
The field of infectious diseases has been revolutionized by advances in PCR and nucleic extraction methods. Compared to traditional serology or culture techniques, PCR tests are capable of detecting extremely low levels of infection. The rapid detection of infectious disease pathogens in blood samples, enabled by RT-PCR, allows for faster, more specific identification, which can aid the therapeutic choices made by the clinician.
The use of extracted DNA and RNA in various applications and research fields is widening every year. With the need for high-throughput analysis and downstream applications such as NGS, automation is making its way into laboratories and facilities.
Automation: Many DNA/RNA isolation kits are now compatible with and can be integrated into automation systems. For speedy and reproducible sample processing, robotic liquid handling systems are used for dilutions and washing steps. These eliminate manual errors and offer walkaway features, opening up time for other experiments. It is important, however, that the kits you choose can also be used in manual applications so as to optimize and troubleshoot in the initial phases of the study.
DNA and RNA co-extraction: To make the most of a sample, kits are being developed where DNA and RNA can be extracted at the same time. These systems alleviate the need for separate kits and save time for downstream processing techniques such as RT-PCR.
DNA barcoding: Similar to barcodes used in grocery stores to identify a product, DNA barcodes are used to identify the species that the DNA belongs to. DNA barcoding has become a routine application in the food industry to test contaminants and to characterize the true source of fish and meat.
Isothermal nucleic acid amplification: Eliminating the need for a thermocycler and controlled temperature fluctuations, isothermal nucleic acid amplification technology — known as recombinase polymerase amplification (RPA) — is making waves. Best suited for field studies and resource-poor settings, RPA is employed by scientists and clinicians alike. Read our interview with a scientist who uses RPA on a ‘mobile laboratory’ as he travels across the world.
Many different types of DNA and RNA purification kits and quantification systems exist in the market. Selecting the correct kit or system for your starting material and downstream application may seem like an overwhelming task, but by applying a few simple considerations, you should find choosing the right kit or system much easier.
Visit the SelectScience® product directory for an overview of the latest DNA and RNA Purification and Analysis technology from leading manufacturers and read user reviews. Keep up-to-date with the latest purification methods by visiting the SelectScience application note and video libraries in life sciences, drug discovery and clinical community.