Many research areas begin with a sample of DNA or RNA — and how those nucleic acids are extracted for study can greatly impact your results. Here we focus on helpful tips from scientists, advice on making the extraction workflow run more smoothly, and products that save time and improve results.
Automation to improve workflow
Extraction is particularly amenable to automation, which can smooth the workflow, and increase reproducibility by reducing variability. Improving nucleic acid workflows begins with looking at all steps involved to identify potential sources of variability. “The sample matrix, purification process, number of human intervention steps, and downstream assays can all contribute to the variability seen in the end results,” says Douglas Horejsh, senior research scientist at Promega. Automation can reduce variability simply by minimizing human involvement in steps such as pipetting.
Promega offers a wide range of products for nucleic acid extraction, including manual and automated solutions. The automated Maxwell® RSC help researchers maximize reproducibility when purifying nucleic acids. “For some products such as our Maxwell RSC ccfDNA Plasma kit, the scientist need only to add 1 ml of plasma and start the run, with no other steps for the user,” says Horejsh. In late 2017, Promega plans to release the MaxPrep™ Liquid Handler, to automate pre-processing steps like Maxwell deck tray set-up and post extraction sample preparation. The Maxprep Liquid Handler and higher-throughput Maxwell RSC 48 are part of a new modular, expandable automation system Promega will be introducing later this year.
QIAGEN also offers a range of kits for isolating nucleic acids, as well as automated solutions on their QIAcube, QIAcube HT, EZ1, or QIAsymphony platforms. Automation is especially helpful “when you deal with samples with inevitable biological variations, such as blood or tissues,” says Peter Porschewski, associate director in R&D at QIAGEN. QIAGEN extraction kits for particular sample types include QIAamp kits for human samples such as serum, miRNeasy kits for microRNAs, or the new DNeasy Power Kits for microbiome studies.
Direct binding to reduce size bias
Innovations in binding chemistry are helping to reduce extraction and purification times. With a patented, direct-binding technology, Zymo Research’s Direct-zol™ RNA Kits use a 7-minute phenol-free protocol to purify NGS-ready RNA from TRIzol®-type reagents. Traditional methods for extracting RNA from TRIzol-type reagents take about 45 minutes, and involve multiple centrifugations and phase separation steps, including organic extraction and alcohol precipitation. “Additionally, because the purification is performed directly on a solid surface, such as a spin-column or magnetic beads, we avoid the selective loss of small RNAs of low GC-content observed during phase separation.” says Ryan Kemp, director of nucleic acid solutions at Zymo Research.
Innovations in binding chemistry are helping to reduce extraction and purification times.
Direct detection in samples
Direct detection of RNA in a sample is possible using Bio-Rad’s SingleShot™ Cell Lysis Kit. Adding the reagent to cultured cells allows researchers to isolate total RNA in less than 10 minutes, according to Yann Jouvenot, marketing manager in the Life Science Group at Bio-Rad Laboratories. This direct lysis buffer lyses cells and stabilizes RNA for subsequent quantitative PCR. This method has the advantage of speed and fewer steps, as opposed to the column-based extraction tools. A shorter workflow helps to isolate RNA, which is more labile and degrades more easily than does DNA. The same lysate can also be tested by western blot.
In addition to direct detection reagents, Bio-Rad Laboratories’ also offers a range of other tools. The AurumTM line of column-based tools supports DNA or RNA extraction using silica membranes. Jouvenot recommends these for extracting highly purified nucleic acids from a variety of different samples. Bio-Rad also offers DNA extraction using resins, such as the Chelex resin in their InstaGene™ Matrix, that bind to PCR inhibitors. Another tool, PureZOL™ RNA Isolation Reagent, can be used to isolate RNA, DNA, and protein from the same sample. “In terms of purity and concentration, PureZOL lysis remains popular because it can work with solid samples such as FFPE tissue,” says Jouvenot.
Choosing the right workflow
To improve nucleic acid extraction, it’s helpful to consider the entire workflow from sample to insight, says Porschewski. The manner in which biological samples are collected and stored, for example, can strongly affect the quality of the results. “It is important to consider stabilizing your sample, using stabilization reagents like RNAlater or complete systems like the PAXgene System, where we offer blood collection tubes to stabilize RNA or circulating DNA in clinical samples,” notes Porschewski.
Above all, it is important to choose the right workflow for the experiment, according to Jouvenot. This choice will be influenced by the nature and concentration of the starting material, and the end product that the researcher is looking for. “We recommend the column-based tools if you need highly purified DNA or RNA, or if you’re looking for concentration, but these workflows involve more steps,” he says. On the other hand, direct detection methods such as resin or direct lysis buffer allow for a more rapid and streamlined workflow.
Tools for active research areas
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New tools for active research areas continue to emerge, such as kits to isolate cancer biomarkers from liquid biopsies (i.e. human samples such as blood, urine, or saliva). Targets include circulating cell free DNA (ccfDNA), circulating tumor cells (CTCs), and exosomes. “The ccfDNA is highly fragmented and the concentration is very low, so it is important to have a highly efficient extraction,” says Porschewski. QIAGEN’s QIAamp MinElute ccfDNA Mini and Midi kits purify ccfDNA from up to 10 ml of serum or plasma. The AdnaTest ProstateCancer CE allows analysis of CTCs. For exosome and exosomal RNA isolation, QIAGEN offers the ExoEasy and ExoRNeasy kits.
The performance of four different DNA extraction methods, a soil DNA extraction kit, and a fecal DNA extraction kit was assessed using the ZymoBIOMICS® Microbial Community Standard. DNA was then subjected to 16S rRNA gene sequencing. The microbial composition was determined by mapping raw sequencing reads against reference 16S sequences of the strains contained in the standard. Image courtesy of Zymo Research.
In the rapidly growing field of microbiomics, workflow variations between Human Microbiome Project laboratories were shown to be responsible for bias in results. Scientists at Zymo Research helped to identify the sources of variation. Sample lysis and nucleic acid extraction were among the steps shown to contribute substantial bias. For example, failure to control for differences in cell lysis susceptibility biased the results toward the easy-to-lyse gram-negative bacteria. “Zymo Research developed and validated a complete workflow using stringent standards to overcome bias and error at every step, including lysis-induced bias,” says Kemp. “The ZymoBIOMICS DNA Mini kit was launched specifically to address lysis efficiency and has been validated for unbiased lysis.” Zymo’s new ZymoPURE EndoZero™ kits extract plasmid DNA with no anion exchange, gravity flow, or precipitation steps — and with endotoxin levels well below industry standards.
Even though many researchers assume that extraction is a “no-brainer,” Kemp urges them to incorporate controls into their workflow. “Microbiome researchers have generated years of data that can’t be reproduced between labs, because many groups didn’t have controls or references in place,” he says. “Using controls and validating workflows as a standard practice is important to prevent that.” The continuing development of extraction tools that contribute to greater reproducibility is making this easier to do.
Images: Dreamstimes Images & Zymo Research