Caitlin Smith has a B.A. in biology from Reed College, a Ph.D. in neuroscience from Yale University, and completed postdoctoral work at the Vollum Institute.
Extracting nucleic acids from tissue samples is commonly used for downstream processes, such as subcloning, library generation, real-time quantitative PCR and traditional Sanger or next-generation sequencing. Kits for the extraction of nucleic acids are valuable tools, because they save time and offer convenience and reliability. Typically, nucleic acid extraction kits include a buffer for tissue homogenization and cell lysis as well as a tool for extracting and collecting the DNA and/or RNA from the lysate. But with so many options, how do you know which kit to choose? A key factor in kit selection is the type or state of the tissue you are working with. Tissue preparations can be obtained as fresh material, frozen or fixed—also known as formalin-fixed paraffin-embedded (FFPE). Certain tissue types may be more difficult to lyse than others, and this can sway the decision to use one method rather than another.
Here we provide an overview and discussion of some of the available options and features of different nucleic acid extraction kits to assist you in performing your experiments.
Many ways to lyse
Lysing a tissue sample is the initial step required to release its nucleic acids. The method you use depends upon the tissue type, as some are more difficult to homogenize and lyse than others, making extraction tricky. Many tissues are typically homogenized in an aqueous lysis reagent that includes buffers such as Tris and EDTA, salts and a detergent such as SDS or Triton X-100. Lysis reagents for RNA extraction also may include RNase inhibitors.
The abundance of contractile proteins in skeletal muscle, connective tissue in heart and collagen in skin makes lysis challenging, requiring specialized reagent formulations. The most common modifications of reagents are high concentrations of chaotropic salts, like guanidine thiocyanate or urea, or the addition of proteases and proteinase K to selectively break down proteins. Bio-Rad, QIAGEN, Thermo Fisher Scientific and other vendors provide special kits that are designed to address the more challenging-to-lyse tissue samples.
Often, very rare or precious material is preserved as FFPE samples. Extracting nucleic acids from fixed tissue requires some extra steps, and there are kits specifically designed to work on FFPE samples, such as Promega’s ReliaPrep FFPE gDNA MiniPrep System. The first step is to remove the paraffin, which may be dissolved using xylene and ethanol washes or mineral oil. After paraffin removal, the tissue must be rehydrated before lysis—and then extraction can be performed.
Sometimes paraffin removal requires multiple washes, which can result in sample loss—a problem for rare or precious samples. If this is a concern, consider kits that require fewer steps for paraffin removal. For example, Analytik Jena’s blackPREP FFPE DNA Kit (and the soon-to-be-released innuPREP FFPE RNA Kit) perform the paraffin removal and lysing steps simultaneously. MoBio Laboratories’ BIOstic® FFPE Tissue DNA Isolation Kit also includes a paraffin-removal reagent that can be used concurrently with lysing. Another method uses heat to melt paraffin and lyse cells—the QuickExtract™ FFPE DNA Extraction Kit from Epicentre (an Illumina company).
Capture and release methods
Most extraction kits take advantage of the ability of silica to bind DNA or RNA. Two ways in which silica is used in kits are in spin columns and in silica-coated magnetic beads. With the first option, a silica-coated membrane fits inside a spin column. Lysate is added to the spin column, and centrifugation drives the lysate through the membrane, leaving the nucleic acids bound to the membrane. The nucleic acids can be released by addition of water or buffer. With the second option, magnetic beads coated with silica collect the nucleic acids, which are then magnetically separated from the rest of the lysate.
A recent improvement to spin-column kits is faster lysis time related to proprietary changes in lysis buffer components. Examples include Analytik Jena’s innuPREP DNA and blackPREP DNA kits. Ingo Jenneckens, export manager for life science at Analytik Jena and Biometra, says this works by using “a combination of chaotropic and antichaotropic salts, allowing the extraction to take place at low salt concentrations as well as low ionic strength.” Such conditions prevent inhibition of enzymes like proteinase K, which in turn shortens lysis time, “especially for complex starting material,” he says. In addition, QIAGEN is about to release a new kit with a faster lysis step called the QIAamp Fast DNA Tissue Kit. This kit introduces “an improved lysis protocol,” says Marco Polidori, global product manager at QIAGEN.
Other spin-column kits have been optimized for RNA extractions. Examples include Promega’s ReliaPrep RNA Cell and Tissue Miniprep System, Roche Diagnostics’ High Pure RNA Tissue Kit, Bio-Rad’s Aurum™ Total RNA Mini Kit, Norgen Biotek’s Animal Tissue RNA Purification Kit and Analytik Jena’s innuPREP RNA Kit. If you need to extract both DNA and RNA from the same starting sample, check out QIAGEN’s AllPrep DNA/RNA Mini Kit and Norgen Biotek’s RNA/DNA Purification Kit. Norgen Biotek uses resin-based columns which, in contrast to most silica-based spin-column systems, “bind all RNA species at an equal rate, irrespective of size,” says Yousef Haj-Ahmad, president and CEO of Norgen Biotek. “And [they] bind the small RNA without bias to GC content.”
Spin columns are convenient and easy to use and are often the choice of labs with lower-throughput needs. For labs that handle or process multiple samples at once, extraction kits that use silica-coated magnetic beads are an option. Silica-coated beads are mixed with a lysate, the released nucleic acids in solution bind to the beads, and a magnetic system is used to “pull” the complex away from the lysate. Adding water or buffer releases the nucleic acids from the beads. The process can be very fast and requires minimal handling, as the magnets are doing most of the work. Magnetic bead-based kits include Corning’s Axygen® AxyPrep Mag Tissue Genomic DNA Kit, and the Mag Tissue-Blood Kit, which can “simultaneously extract and purify DNA from tissue and blood samples,” says Julie Mai, product line manager at Corning Life Sciences.
Chemical methods
Some kits don’t use silica, but rely instead on chemistry inside the sample tube. For an inexpensive alternative to extraction kits, consider the DNAzol® Reagent from Life Technologies (part of Thermo Fisher Scientific). Tissues are homogenized and lysed in DNAzol, a guanidine-detergent-based organic reagent, and cellular debris is pelleted and removed by centrifugation. DNA is then precipitated by addition of ethanol. Roche Diagnostics also offers a one-tube protocol with its DNA Isolation Kit, which uses a lysis buffer containing a strong anionic detergent and proteinase K. After homogenization and lysis, RNA is removed by adding RNase to the tube, and proteins are precipitated, then pelleted and removed by centrifugation. The DNA left in solution can be precipitated by ethanol addition.
Another single-tube lysis and extraction solution is the KAPA Express Extract from Kapa Biosystems. In this kit, the lysis reagent contains a thermostable protease for heat-lysing the cells, thereby releasing nucleic acids into solution. This is especially useful when extracting nucleic acids to use as PCR templates—simply pipette the DNA extract into your PCR reaction mixture. This is often used for genotyping from a variety of tissues, according to Maryke Appel, technical director at Kapa Biosystems: “The combination of rapid extraction and efficient, crude sample PCR represents a unique solution for fast genotyping and other qPCR applications.”
Automated extraction
Several vendors provide automation-friendly versions of their extraction kits for increased throughput using 96-well plate formats. Options for fresh or frozen tissue include Life Technologies’ PureLink 96 Genomic DNA Kit and ChargeSwitch® Direct 96 gDNA Kits (from Thermo Fisher Scientific) and Roche Diagnostics’ MagNAPure Kits. Charles Hardwick, senior field applications specialist in technical operations in life sciences at Roche Diagnostics, says automation can help make results more consistent. “The main challenge for nucleic acid extraction is reproducibility,” he says, but robotic platforms such as those offered by Roche “are ‘walk away,’ and guarantee sample-to-sample and run-to-run consistency.”
For FFPE tissue samples, the truXTRAC™ system from Covaris uses acoustic energy to remove paraffin and rehydrate tissue; the system then purifies nucleic acids with magnetic bead-based methods or spin columns. “Designed specifically for FFPE samples, the truXTRAC process utilizes highly controlled, focused acoustic energy for the active removal of paraffin from FFPE sample while simultaneously rehydrating the tissue,” says Guillaume Durin, senior product manager at Covaris. And although most conventional paraffin-removal methods use tissue slices 5 to 10 microns thick, Covaris’ method “is not as limited by the thickness of FFPE tissue and enables extraction of high-quality nucleic acids.”
A plethora of nucleic acid extraction kits is available, and the choice can be overwhelming. Following these tips on the various lysis and extraction methods will have you isolating pure and clean nucleic acid samples. Those next experiments involving cloning and genomic characterization will be a breeze.
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