by Caitlin Smith
Extracting and purifying RNAfrom cells or tissues is no easy task, as anyone needing pure RNA for gene expression analysis will tell you. One of the biggest problems is protecting RNA from ubiquitous enzymes that would otherwise degrade it rapidly. But the challenges don’t stop there. Optimizing the many parameters involved in RNA purification—such as high yield, reproducibility, speed, throughput and quality—can be a full-time job in itself. That’s why so many researchers now turn to previously optimized RNA purification kits. Specialized protocols, such as those for isolating mRNA or microRNA (miRNA) or extracting RNA from formalin-fixed paraffin embedded (FFPE) tissue, present new sets of challenges. Today’s optimized RNA extraction tools are surely welcome.
Homogenization and stabilization
Quality RNA results begin with an RNase-free environment. “Researchers need to be aware that RNase contamination and RNA degradation are still their worst enemies, even though the RNA preparation technologies have improved,” says Nathan Harris, product manager at Ambion, of Life Technologies. “It is still a good idea to be ‘paranoid’ about RNases and focus on creating an RNase-free environment. The surest way to do this is to work exclusively with RNase-free tips, plastics and reagents. In addition, the lab space must be wiped down with an appropriate chemical to remove RNases.”
One might think that sample homogenization, to release RNAs from cells, is the first step in preparing RNA. However, a crucial step precedes it: ensuring your sample is stabilized upon tissue disruption right after sample collection, when the RNA immediately begins to degrade. It’s wise to use a solution that can stabilize your RNA upon its release from cells. “RNA is extremely labile and susceptible to degradation by RNases,” says Janina Lehmann, director of sample technologies business at Qiagen. “RNA integrity must be preserved following sample disruption and homogenization. To prevent RNA degradation and changes in the transcript level, Qiagen recommends stabilization of RNA immediately after collection, for example by using the RNeasy Protect Mini Kit, a one-kit solution enabling sample stabilization and purification of RNA.” Qiagen provides a range of homogenization tools for different sample types, from the QIAshredder spin columns, for fast and simple homogenization of cell lysates, to the TissueRuptor and TissueLyser systems, for mechanical disruption and homogenization of tougher samples.
Another factor to keep in mind is time—the faster the better. “Immediate RNA stabilization after collection and efficient sample lysis, and RNase inhibition, are essential to maintain RNA integrity,” says Lehmann. “Up- or down-regulation of genes and enzymatic degradation of RNA can result in a transcription profile that is considerably different from the actual profile in the in vivosample. These changes can occur very rapidly, during sample collection and handling and before sample lysis, and can inhibit further changes in the gene expression pattern.”
Other important aspects of sample collection include storage and temperature. Larger samples, like whole organs, require more processing time (during which RNases go to work), and samples may need to be stored prior to homogenization. “Collecting intact organs and freezing them immediately ensures better results than cutting up and damaging a tissue,” says Harris. “If a cold environment is not available, then a stabilization solution such as RNAlater® is essential. Working fast at a reduced temperature is the best way to ensure that you get high-quality RNA.”
To take full advantage of current methods of RNA preparation, Marie-Louise Lunn, director of product management at Exiqon, suggests learning the principles of optimization by preparing RNA “from scratch” (i.e., without a kit); it’s an educational experience that can be useful when subsequently prepping RNA with or without kits. “Trying conventional RNA isolation methods helps build an understanding of the RNA extraction principles and process,” says Lunn. “Whether using a kit or a conventional procedure, understanding the RNA extraction process is important for knowing when to be very cautious in your protocol, and when a step is less critical if not performed optimally.”
Though some cells are disrupted during homogenization, a subsequent cell lysis step ensures that as much RNA is released from cells as possible. Harris notes that the crucial step of counting cells correctly can have dire repercussions if it’s inaccurate. “Experiments have been ruined because [scientists] thought that they were working with 100,000 cells, when in reality they were working with one million,” says Harris. “This type of oversight can result in failed lysis or clogged purification columns, and no RNA at the end of the preparation.”
Special additives may be beneficial at the cell lysis stage, to manage the contents released from the particular types of cells you are working with. For example, Exiqon recently introduced its Lysis Additive, which is useful when extracting RNA from samples with high lipid content, such as brain tissue. “An increasing amount of miRNA research is performed in neuronal samples, hence based on high market demand and requests from our customers, we now offer a protocol specifically for fatty tissue such as brain,” says Lunn. “It is necessary to cautiously consider [whether] the sample composition matches the extraction procedure one uses. Do you need to perform some pre-processing of the sample before engaging in the actual RNA isolation protocol? This relates to any type of RNA isolation protocol used. As an example, certain RNA samples may be impeded by high concentrations of polysaccharides (e.g., hyaluronic acid in vitreous humor). This may require an upfront enzymatic degradation step to reduce the viscosity and allow efficient isolation afterwards (e.g., by column-based RNA isolation protocol). However, such additional steps should be weighed against the potential disadvantage of overall loss of RNA due to endogenous RNases.”
Roche’s RealTime Ready Cell Lysis Kit is designed for cell lysis—and for circumventing the subsequent purification step. It is used to prepare cultured cells directly for real-time RT-PCR for rapid gene expression analysis. “This kit uses a five minute modified, one-step protocol to instantaneously lyse up to 30,000 cells,” says Burkhard Ziebolz, head of global communication at Roche Applied Science. “The resulting lysate is used directly for cDNA synthesis. There is no need to perform the time-consuming RNA purification step. Resulting high-quality cDNA can be analyzed using real-time PCR experiments. Very high throughput, cellular RT-qPCR studies are possible.” The kit can be adapted to integrate into a researcher’s manual or automated gene expression workflow.
Extraction and purification
The extraction and purification steps of RNA preparation are the heart of commercial RNA prep kits, and with good reason. The kits have been meticulously developed to work optimally by simply following the protocol. Hopefully, this saves you most or all of the painstaking optimization process. Kits today are diversifying, so you can save even more time by choosing a kit that has been optimized for the type of sample you are working with and/or the application you are performing.
High throughput:Two manufacturers have recently optimized their systems for high-throughput preparation. Roche’s MagNA Pure 96 Automated Sample Preparation Instrument and MagNA Pure 96 Cellular RNA Large Volume Kit are designed for high speed and high throughput. “The RNA isolation procedure is based on the proven MagNA Pure Magnetic Glass Particle Technology,” says Ziebolz. “The dual-head robot prepares 96 RNA samples in less than 90 minutes. MagNA Pure 96 purification is straightforward for a broad range of sample materials, including whole blood, cultured cells, FFPE tissue, fresh-frozen tissue, RNAlater-stabilized tissue and PAXgene Blood RNA tubes. MagNA Pure 96 purification reagents are prefilled in sealed containers that are easily positioned on the instrument stage, minimizing pipetting and contamination risk.”
Additional high-throughput kits are offered by Ambion, of Life Technologies, with two kits designed to extract nucleic acids from FFPE samples. The MagMAX FFPE Total Nucleic Acid Isolation Kit can recover all RNA types (from microRNA to ribosomal RNA) as well as (if you choose) genomic DNA. The MagMAX FFPE DNA isolation kit is designed to recover only genomic DNA. “What makes these products different is the MagMAX™ magnetic bead technology,” says Harris. “This technology provides scientists with the flexibility to scale up their nucleic acid extractions from 96 FFPE samples [in three hours] without using toxic chemicals. Nucleic acid isolation from FFPE is currently viewed as rigid and not compatible with high-throughput platforms. We believe the MagMAX FFPE kits will change that viewpoint. The best part is that there is no compromise on nucleic acid yield or purity. These kits were benchmarked against our highly regarded Ambion® RecoverAll™ Total Nucleic Acid Isolation Kit for FFPE, so the end user can expect the same kind of results, just at a larger scale.”
MicroRNA from total RNA:Exiqon offers two RNA isolation kits for extracting total RNA from a broad variety of sample types. One kit is specific for RNA isolation from cells and plant material, including whole blood, bacteria, yeast and fungi. The other kit is designed for optimal total RNA isolation from animal tissue. Although some companies make separate kits for mRNA or microRNA (rather than total RNA), Lunn believes it is better to isolate only the total RNA. “Our RNA isolation kits are designed to extract total RNA, including the microRNA content, in the samples,” says Lunn. “Even when only the microRNA content is required, it is in fact very important that the isolation kit prepares total RNA. This will better reflect the actual microRNA content in the samples. Adding additional steps in the RNA extraction protocol for specific microRNA cleanup (typically afterwards) in the process causes additional manipulation to the samples and thus the risk of introducing bias to the actual microRNA content.” Exiqon’s total RNA extraction kits are designed to work optimally with its downstream applications, including products for microRNA research such as microRNA microarrays and microRNA qPCR.
Quality checks for microRNA:Another new RNA prep tool from Exiqon is the RNA Spike-in Kit for checking the quality of Extracted RNA. The kit is designed to work with the company’s RNA isolation kits and its downstream microRNA qPCR system. “Adding RNA Spike-ins (which are synthetic microRNAs) in controlled and known concentrations to the sample during the RNA isolation procedure makes it possible to check the extraction efficiency of the microRNA content, which is very difficult to otherwise monitor and measure,” says Lunn. This important information can save you from working on poor-quality samples in downstream qPCR experiments. The company is in the process of launching a quality-control qPCR panel for testing RNA samples prior to running large microRNA qPCR studies.
MicroRNA isolation from small samples:An additional technology for purifying RNA is the basis for Qiagen’s RNeasy line of RNA kits. These kits incorporate a specialized lysis buffer containing guanidine salt for RNase denaturation and a silica membrane that binds RNA specifically. After the lysate is washed off, pure RNA stays on the membrane and can be eluted. “Purification of total RNA or miRNA requires optimized binding conditions to the silica membrane, as provided by various optimized RNeasy Kits,” says Qiagen. [Should this be attributed to Lehmann, of Qiagen?] “For isolation of miRNA from small sample volumes of cells and tissue, we have recently introduced the miRNeasy Micro Kit.” This kit provides high-quality, concentrated miRNA for reliable and reproducible results in downstream applications, without the need for subsequent concentration or cleanup.
All-in-one purification of DNA, RNA and protein:In addition to a wide range of RNA kits for different sample types, Qiagen offers all-in-one kits. “For simultaneous purification of RNA, DNA, and protein, Qiagen offers the AllPrep product line for maximal recovery of each analyte in a single procedure,” says Lehmann. “Depending on the sample type, we offer dedicated kits; for example, the Allprep DNA/RNA FFPE Kit is specially optimized for purification from FFPE tissues.”
The present and the future of RNA preparation
It’s no surprise that a method as widely used as RNA preparation is evolving quickly. Commercial kits are becoming more specialized to meet growing demands. "In recent years, more kits [have been] designed for RNA isolation from samples of specific origins or for isolating specific proportions of the RNA,” says Lunn. “However, these kits may not always be suitable for the downstream applications. Hence, the challenges lie in developing kits for specific sample types tailored to specific downstream applications, where quality is not compromised and content still reflects the actual RNA without having introduced any bias.”
Looking toward the future, Harris notes that the definition of RNA preparation is changing. “People are moving away from thinking that RNA prep is solely ‘binding, washing and eluting,’” he says. “Crude lysate technologies, such as the one in our Cells-to-CT™ product line, are allowing researchers to get the same results in a faster, more cost-effective way. As this shift in thinking continues, one can imagine that RNA will be ‘prepared’ and analyzed in vivo, eliminating the need for what we think of as traditional RNA prep.”
The image at the top of this page is data from Exiqon's miRCURY™ RNA Isolation Kits.