"Purely RNA:

New innovations enhance the quality, speed, and efficiency of RNA isolation techniques."

By Deborah Stull and Jessica M. Pisano

Deborah Stull (zebrafish9@yahoo.com) is a freelance writer in Lansdale, PA
Jessica M. Pisano (jpisano@fas.harvard.edu) is a freelance writer in Boston, MA

Fundamental laboratory techniques such as Northern blot analysis, RNA protection assays, in situ hybridization, and reverse transcriptase-PCR (RT-PCR) require high-quality, highly purified RNA samples. Preparing such samples is often laborious at best, because RNAses-both stable and omnipresent-can easily degrade the samples. In the past, RNA work was often left to dedicated labs, with dedicated work areas, equipment, and some very meticulous workers. Fortunately, the technology continues to evolve, and numerous RNA-purification protocols now exist that allow researchers to focus on specific scientific questions instead of on sample quality. These improvements are making RNA isolation more routine, enabling labs that lack RNA-handling expertise to venture into the arena of gene expression analysis.

The first step to RNA recovery is cell lysis, which depends on the denaturation of the proteins that comprise the cellular membrane. In 1979 John Chirgwin and colleagues devised a method for disrupting cellular membranes without denaturing nucleic acids. They homogenized tissue in guanidinium thiocyanate, a protein
denaturant, and b-mercaptoethanol, a reducing agent.[1] These scientists then isolated and purified the cellular RNA through either ethanol extraction or ultracentrifugation across a cesium chloride gradient. This technique was the first to allow researchers to isolate RNA specifically, but it had a number of drawbacks-it was lengthy, inefficient, hazardous, and inconsistent.

In 1987 Piotr Chomczynski and Nicoletta Sacchi improved on this method by combining the extraction procedures into one step through the use of a mixture of guanidinium thiocyanate and phenol-chloroform.[2] This modification had at least two tangible benefits. First, it reduced the length of the RNA-isolation step, allowing researchers to increase the number of samples they collected at a time. This one-step procedure also reduced RNA loss, enabling scientists to purify RNA from smaller sample sizes. In spite of these advances, the method still suffered from shortcomings. For example, ultracentrifugation not only requires expensive instrumentation but also frequently creates RNA pellets that are difficult to resuspend. In addition, extraction with harsh organic solvents can lead to the loss and fragmentation of the RNA sample.

New improvements to these methods, offered by suppliers ranging from Ambion Inc. of Austin, Texas, to Zymo Research of Orange, Calif., simplify and streamline the RNA-isolation process. For example, Ambion's ToTALLY RNA™ TOTAL RNA Isolation kit, based on Chomczynski and Sacchi's guanidine thiocyanate, phenol:chloroform principle, includes all necessary reagents except isopropanol. According to Ambion, this kit produces high-quality RNA from 10 g of tissue or 10⁹ cultured cells from a wide variety of sources including plant tissues and bacteria.

Amersham Biosciences of Piscataway, N.J.; CPG Inc. of Lincoln Park, N.J.; and Maxim Biotech Inc. of San Francisco also offer RNA isolation kits that utilize the one-step guanidine thiocyanate and phenol:chloroform extraction method. According to Maxim Biotech, scientists can use the company's Gstract™ Total RNA Isolation Kit to produce total RNA from virtually any tissue or cell type. The kit's protocol includes an
additional extraction step to eliminate DNA and other contaminants so that the RNA isolated can be used directly in applications such as RT-PCR.

Amersham Biosciences' QuickPrep Total RNA Extraction Kit also takes advantage of the destructive properties of guanidine thiocyanate, but it uses lithium chloride and cesium trifluoroacetate (CsTFA) for selective precipitation and isopycnic centrifugation.[3] This procedure does not rely on organic solvents and can produce RNA from sample sizes ranging from 25 mg to 1 g or 10⁶-10⁸ cells in 60 minutes using an ordinary microcentrifuge. In addition, centrifugation in CsTFA affords a high degree of protection from RNAses.

Recently, some companies released nonhazardous alternatives to the guanidinium thiocyanate/phenol- chloroform mixtures described above. Firms such as PGC Scientifics Corp. of Frederick, Md., offers RNA NOW®, a biodegradable, environmentally friendly, phenol- free solution that can isolate RNA from 10⁷ cells or 100 mg of tissue in 60 minutes. Qbiogene Inc. of Carlsbad, Calif., also offers researchers an alternative to the use of guanidine thiocyanate and organic solvents. The company's Total RNA Safekit™ is designed to process 10-100 mg tissue samples to yield up to 100 µg of total RNA that is ready, according to Qbiogene, for applications such as differential display and RT-PCR.

Out for a Spin

Several companies have modified the guanidinium lysis method to replace the organic solvent-based extraction steps with RNA-adsorbing silica- or glass-based spin-columns.[4] The primary advantage to these systems is that users avoid the use of hazardous organics such as phenol, chloroform, and isoamyl alcohol. Depending on how the system is devised, spin-column-based RNA purification protocols force the cell lysate through the filter by either centrifugation or under vacuum. For small-scale preparations, companies can house these filters in microcentrifuge tubes to simplify their use.

Valencia, Calif.-based QIAGEN's RNeasy® Stabilization and Total RNA Isolation System is one spin-column-based system. The kits in the RNeasy line produce high-quality RNA from animal cells and tissues as well as plant and fungal cells and tissues. Using the RNeasy system, investigators can collect samples in as little as 30 minutes without phenol/chloroform extraction, centrifugation through a cesium
chloride gradient, or lithium chloride or alcohol precipitation. QIAGEN also offers RNeasy Protect Kits for animal tissues and RNeasy Protect Bacteria Kits for Gram-positive and Gram-negative bacteria. These kits include the RNAlater RNA stabilization reagent, which immediately stabilizes the cellular RNA profile so that downstream applications accurately reflect the expression status at the time of harvest.

Hombrechticon, Switzerland-based PreAnalytiX, a joint venture between QIAGEN and BD Biosciences, recently introduced the PAXgene™ Blood RNA System, which clinical researchers can use to collect, store, stabilize, and isolate RNA from whole-blood samples. Phlebotomists draw blood into specialized PAXgene Blood RNA Tubes that contain a stabilizing reagent that permits blood samples to be stored for up to five days at room temperature. Researchers then isolate the RNA using the PAXgene Blood RNA Kit; like QIAGEN's RNeasy kits, this system purifies RNA using a spin column.

Indianapolis-based Roche Molecular Biochemicals' High Pure™ RNA Isolation Kit purifies RNA using a glass fiber fleece in a microcentifuge filter tube. The kit employs a single reagent to lyse cells and inactivate RNAses, and the fleece binds the total RNA selectively during DNAse treatment and DNA removal. According to Roche, this kit can be used with sample preparations as diverse as human whole blood and
yeast, and allows researchers to purify total RNA from a number of samples in minutes.

Stratagene of La Jolla, Calif., offers the Absolutely RNA™ Nanoprep Kit, which allows researchers to prepare RNA from as little as a single cell. After cell lysis, the lysate is applied to a silica-based fiber matrix that is housed in a nano-spin cup to bind the RNA during the DNAse treatment and DNA removal steps. With an elution volume of only 10 µl, the resulting RNA is sufficiently concentrated to be useful for applications such as quantitative RT-PCR and the preparation of fluorescent microarray targets.

Stratagene also offers researchers the Absolutely RNA RT-PCR Miniprep Kit for up to 10⁷ cells or 40 mg of tissue and Absolutely RNA Microprep kit for up to 5 x 10⁵ cells, useful for laser microdissection samples. Finally, the company produces a StrataPrep®-96 Total RNA Purification Kit, which is useful for high-throughput RNA purification. The StrataPrep-96 kit permits researchers to prepare RNA samples in
96-well microtiter plates that are compatible with most common 96-well plate vacuum manifolds and centrifuge rotors.

The majority of RNA isolation kits require either fresh or frozen tissue or cells as starting material. However, these samples are not always immediately available, as tissues of interest are often scarce and need to be used for multiple applications. To address this problem, Zymo Research developed the Pinpoint Slide RNA Isolation System. This system allows researchers to easily isolate total RNA directly from
selected areas of tissue sections on microscope slides. Scientists can use the Pinpoint Slide Isolation System I to recover total RNA of interest from fresh or frozen tissue sections fixed in a variety of solutions, and can use the Pinpoint Slide Isolation System II to purify RNA from selected sections of paraffin embedded tissue.
Researchers can therefore examine and document rare tissue anatomically and then collect and analyze RNA specifically from those regions of interest.

The Matrix

Often the isolation of total RNA is just the first step in the purification of mRNA. The most common procedure for the isolation of mRNA relies on base pairing between the 3' poly(A)-sequence terminating eukaryotic mRNA molecules and a synthetic chain of 20-30 thymine residues, called oligo[dT]. Suppliers usually covalently link the oligo[dT] chains to a cellulose matrix via the oligo's terminal 5' phosphate. Users can then purify mRNA either by batch binding and washing in an oligo[dT]-cellulose matrix suspension or by gravity-driven column chromatography.

Ambion offers several Poly(A)Purist™ mRNA Purification Kits based on oligo[dT]-cellulose selection: the Poly(A)Purist™, the MicroPoly(A)Purist™ small-scale mRNA purification kit, and the Poly(A)Purist Mag mRNA purification kit. These kits use batch binding of RNA to pre-measured aliquots of oligo[dT]-cellulose to circumvent the problems frequently associated with conventional column technology, including slow flow rates and clogged columns. The Poly(A)Purist Mag kit uses magnetic bead-based selection (see below). The mRNA purified with these kits can be used in Northern blot analysis, RNA protection assays, and RT-PCR.

Amersham Biosciences also offers a variety of kits that use oligo[dT]-cellulose columns for mRNA purification. The mRNA Purification Kit purifies mRNA from total RNA extracted from small samples in just 30-45 minutes, according to the company. The kit includes pre-dispensed reagents, which reduces the risk of nuclease contamination.

Both BD Biosciences-CLONTECH of Palo Alto, Calif., and QIAGEN offer alternatives to oligo[dT]- cellulose matrices. CLONTECH's NucleoTrap® mRNA Kit and QIAGEN's Oligotex® mRNA Purification System both employ oligo[dT] chains that are covalently bound to latex beads instead of cellulose. The perfectly spherical surface of the latex beads allows for uniform dispersion and minimal centrifugation time, yielding increased recovery of purified mRNA.

Although most mRNA purification kits rely on extraction of total RNA as a first step, Amersham Biosciences and QIAGEN offer kits that can be used to purify mRNA directly from animal tissues and cells. For example, scientists can use QIAGEN's Oligotex Direct mRNA System to purify mRNA directly from animal tissues and cells. Amersham Biosciences' QuickPrep mRNA Purification and QuickPrep Micro mRNA Purification Kits, also work without previously isolated total RNA. Researchers can use the QuickPrep mRNA Purification Kit to prepare mRNA from 0.5 grams of tissue (5 x 10⁷ cells) in under one hour, whereas the QuickPrep Micro mRNA Purification Kit isolates nucleic acids from 0.1 grams of tissue (1 x 10⁷ cells) in only 15 minutes per sample. Neither of these kits uses oligo[dT]-cellulose columns.

Animal Magnetism

Advances in oligo[dT] technology have resulted in increased yield and purity of mRNA, but some researchers still find the spin columns inconvenient to use. Fortunately, companies such as CPG, Ambion, and Roche provide researchers with an alternative, offering mRNA purification kits based on magnetic separation technology.

Magnetic separation allows mRNA to be purified in a single tube by application of a magnetic field, thus eliminating potential sample loss during liquid handling. The technique is also fast, because centrifugation steps are avoided. Magnetic separation technologies rely on supermagnetic particles that are made from polystyrene or iron oxide plus polysaccharides. These particles become magnetic only when a magnetic field is applied to them. They therefore can be repeatedly separated and resuspended without magnetically induced aggregation.

For mRNA isolation, these magnetic beads are coupled to oligo[dT] molecules via functional groups that coat the particles. Once the poly(A) tail at the 3'-end of eukaryotic mRNAs attaches to the molecules, the user collects the conjugated magnetic particles by applying a magnetic field, and elutes the purified mRNAs from the beads. The purified mRNA is then ready to use.

CPG offers the MPG® mRNA Purification Kit, specifically for the isolation of mRNA from purified total RNA in 15 minutes using magnetic separation technology. The MPG (magnetic porous glass)-streptavidin complex provides a large surface area-to-volume ratio for greater oligo[dT] binding, resulting in greater mRNA recovery. The company also offers MPG Direct mRNA Purification kits to isolate mRNA directly
from animal or plant tissues or cells, without the initial isolation of total RNA.

As mRNA isolation is often just the first step in such applications as RT-PCR, Northern blotting, and dot blot hybridizations, Roche offers the MagNA Pure LC mRNA Isolation Kits I and II for tissue and cells as a fully automated alternative to the magnetic separation method of purifying mRNA. These kits include all necessary reagents and are designed for use with the MagNA Pure LC instrument, which combines nucleic acid isolation with PCR set-up. It automatically fills LightCycler capillaries, as well as any other PCR reaction vials, with purified nucleic acids and PCR master mixes, thus reducing the risk of RNase contamination and sample loss, and ultimately saving researchers' time.

The Hybond Atlas RNA Purification and Labeling Kit from Amersham Biosciences combines RNA purification with cDNA probe synthesis and labeling in a single protocol. First, users isolate total RNA from as little as 10 mg of tissue or 10⁵ cells. The resulting total RNA is then mixed with biotinylated oligo[dT], which binds to the poly(A) sequence of mRNA molecules, and magnetic streptavidin beads to bind to the
biotin. Subsequent application of a magnetic particle separator purifies the mRNA, which is then ready for probe synthesis.

The proliferation of RNA isolation and purification systems is a boon for life scientists. Freed from technically difficult, hazardous protocols, they are now free to concentrate on the use of RNA samples, rather than on their preparation. New technologies allow researchers to isolate pure RNA from smaller and smaller sample sizes, and pre-dispensed and individually packaged reagents offer researchers convenience and speed without sacrificing quality. The wide variety of available options means that researchers can surely find a system to meet their needs, whether they prefer traditional methods of denaturation and extraction, or fully automated methods that integrate RNA isolation with downstream applications. The bottom line is that now any lab-whether focused on RNA or not-can conduct gene expression studies. 

    1. J.M. Chirgwin et al., "Isolation of biologically active ribonucleic acid from sources enriched in
    ribonuclease," Biochemistry, 18: 5294-9, 1979.

    2. P. Chomczynski, N. Sacchi, "Single-step method of RNA isolation by guanidinum
    thiocyanate-phenol:chloroform extraction," Analytical Biochemistry, 162: 156-9, 1987.

    3. H. Okayama et al., "High-efficiency cloning of full-length cDNA: Construction and screening of
    cDNA expression libraries for mammalian cells," Methods in Enzymology, 154: 3-28, 1987.

    4. B. Vogelstein, D. Gillespie, "Preparative and analytical purification of DNA from agarose,"
    Proceedings of the National Academy of Sciences, 76: 615-9, 1979.