Ode to Oligo(dT): Oligo(dT) Takes on a Variety of Faces in Kits for the Purification of mRNA

Date: May 25, 1998 Author: Laura DeFrancesco Charts How many different ways are there to isolate messenger RNA (mRNA)? You can bind it to oligo(dT) cellulose in a slurry, bind it to oligo(dT) on columns, bind it to oligo(dT) attached to latex beads, or bind it to oligo(dT) on magnetic particles. Get the picture? Oligo(dT) is clearly the key to mRNA isolations. And why is that? Considering that mRNA is only one to two percent of the total RNA of a cell, and to make matters worse, is heterogeneous in size, without oligo(dT), and the polyA tails on the messages to which it binds, it's hard to imagine how mRNAs would ever be isolated. In fact, before oligo(dT) came into being, only messages that were either very abundant, like ß-globin in reticulocytes, or very unusual, like the messages for silk fibroin proteins that have an unusual base sequence, had been isolated to any purity at all. In the 26 years since H. Aviv and P. Leder first reported using oligo(dT) to isolate mRNA's ( Proceedings of the National Academy of Sciences, 69:1408-12, 1972), nothing better has come along, though you no longer have to make your own oligo(dT) as they did. But oligo(dT) is not the whole story. Isolating intact RNA is a challenge due to the ubiquity of ribonucleases (RNases). Therefore, equally important as the method for selecting message species is the preparation from which the selection is to be made. While a number of protocols exist (Maniatis' "Cloning Bible" lists five with a couple of variations thrown in), a commonly used strategy is to combine, in a single step, cell or tissue homogenization with the inactivation of RNases using powerful protein denaturing agents. Guanidinium isothiocyanate (GTC) used in combination with ß-mercaptoethanol, for example, both tears cells apart and irreversibly denatures proteins. While originally applied to the isolation of RNA from tissues with high levels of RNase (J.M. Chirgwin et al., Biochemistry, 18:5294-9, 1979), this method has found widespread use because of the high yields and high quality of the RNA obtained. However, while oligo(dT) may be the only game in town, GTC is not. In an attempt to get away from caustic or toxic agents, some companies that make kits for mRNA purification have devised other one-step formulations, incorporating compounds that have been found to do a creditable job of inhibiting RNases. Some of the newer products in the market also streamline the procedure to allow for high throughput applications. (In the original GTC, single step protocol, the lysate was subjected to a long centrifugation step through a dense cushion to capitalize on the fact that RNA is denser than other macromolecules--a procedure bounded somewhat by time and rotor space.) Yield, and Purity Two basic types of kits for mRNA purification exist: the so-called direct kits that isolate mRNA directly from tissue or cells, and indirect kits that work with total RNA (already purified) preparations. The direct kits provide the materials for preparing a lysate from which, for the most part, mRNA can be directly pulled out--that is, no further steps, extractions or precipitations are required once the lysate is made. Indirect kits, on the other hand, contain oligo(dT) and the appropriate buffers, and generally are less expensive than direct kits. While you may be able to save some money with these kits by preparing your own extraction buffers, there also might be times when having clean, prepared solutions is worth the extra expense for the savings in time. The second major decision that has to be made in choosing a kit is the format for the oligo(dT) which, as mentioned earlier, comes in various guises. Kits exist for those of us who like to stick with the tried and true--the oligo(dT) cellulose for gravity column chromatography. In some kits, the oligo (dT) has been pre-measured or put into a tablet to facilitate the preparation of the column and to provide some consistency from sample to sample. However, if the mention of oligo(dT) chromatography conjures images of sitting in front of a column watching drops slowly emerge from the end of a syringe, (with the added excitement of trying to keep the SDS from precipitating out of solution), you might want to look at the kits that use batch isolation or spin columns, which can speed up the process. This isn't just a plus for researchers, but also can improve the quality of the RNA preps by reducing the time of exposure to RNases or other harmful chemicals. The latest innovation in the mRNA purification arena is the introduction of oligo(dT) attached to beads--magnetic or latex. While beads are thought to offer the speediest isolations, some companies that have stuck with oligo(dT) cellulose counter that the yields of mRNA with beads are inferior to oligo(dT) cellulose. In addition, magnetic separation requires special equipment, which can be costly. But bead makers have responded to these charges by increasing the available surface area to up yields of mRNA, and by providing economical solutions for trapping the beads. What follows are short descriptions of 48 kits from 23 companies and a series of tables with some details. The companies have been divided into groups according to the format for oligo(dT), cellulose versus beads, rather than by isolation procedure, since a company is more likely to offer both direct or indirect kits, than different formats for the oligo(dT). One final caveat. Information on yield is provided in some of the kit descriptions. It is important to keep in mind, though, when looking at these figures, that yield will vary not simply with the nature of the isolation procedure, but also with the biological material itself, not to mention the skill of the experimenter. Ambion Ambion makes a variety of products to assist in the preparation of mRNA. Two kits, Poly(A)Pure™ and MicroPoly(A)Pure™ mRNA Purification Kit are designed for the rapid purification of mRNA directly from cells or tissues. These kits utilize a guanidinium thiocyanate detergent solution during homogenization, and because mRNA in the cell lysate is batch bound to oligo(dT), the procedure does not require prior purification of total RNA. Premeasured amounts of oligo(dT) are provided for batch binding --and washing; collection is performed on spin columns for rapid purification. Company tests have shown that polyA tails as short as eight bases long are captured by its material. In addition, the company claims that its oligo(dT) gives yields in the range of 60µg per gram of tissue. In addition, Ambion provides poly(A)+ RNAs from 10 organs of both mouse and rat, and two of human organs. The poly(A)+ RNA is isolated from total RNA using Ambion's Poly(A)Pure mRNA Purification Kit and two rounds of oligo(dT) cellulose selection to minimize rRNA. Other Ambion products include RNaseAlert™, a dipstick assay for detecting RNase contamination of solutions, RNAseZap, an RNase decontamination solution and AntiRNase and placental Rnase Inhibitor. Amersham Pharmacia Biotech Amersham Pharmacia Biotech offers QuickPrep and QuickPrep Micro mRNA Purification Kits which work with a wide variety of eukaryotic cells and tissue samples. The patented method for isolating mRNA directly from tissues and cells on cellulose-(dT) features specially developed pre-packed spun-columns containing oligo(dT) cellulose. These columns circumvent the problems of slow flow rates and clogged columns often experienced during gravity-driven chromatography on oligo(dT)-cellulose. As a result, each cycle of chromatography can be completed in 30 to 45 minutes, according to the company. Amersham Pharmacia's Kits do not require purification of total RNA. mRNA integrity is preserved by utilizing guanidinium thiocyanate early on during the purification. The main differences between the two kits are the number of purifications, the upper limit on the amount of starting sample, the time required to purify mRNA, and the prepackaging of the purification components. AMRESCO The Rapid mRNA Purification Kit can be used to isolate poly(A)+ mRNA from total RNA in 20 minutes utilizing oligo(dT) technology. Batch processing of samples has simplified the procedure, and conserves reagents. Each kit contains all the reagents needed for 25 poly(A)+ mRNA purifications using up to 500µg of purified total RNA as starting material. Company estimates show that purifications can be performed in 20 minutes, using a standard laboratory microcentrifuge. Biotecx Biotecx's ONTrack® is for mRNA purification directly from tissues or cells. Based on oligo(dT)-cellulose spin column chromatography, and using the RNA extraction reagent Ultraspec™ , high yield undegraded mRNA can be isolated in 45 minutes directly from 10 mg to 4.5 g tissue or from 103 to 4.5 x 108 cells. Biotecx's EXPRESEP® Kit isolates in high yield undegraded total RNA in one hour and/or mRNA from purified total RNA in 45 minutes, using oligo(dT)-cellulose spin column chromatography. A set of RNase free buffers is available separately for regeneration and repeated use of oligo(dT)-cellulose columns provided in the EXPRESEP and ONTrack Kits. CLONTECH mRNA Separator Kit contains prepacked oligo(dT)-cellulose spin columns that minimize the handling of RNA samples and maximize the speed and efficiency of washing and eluting the samples. The company reports that 95 per cent enrichment is routinely obtained after only one round of purification with the mRNA Separator Kit, resulting in 20µg to 60µg of poly(A)+ mRNA per mg of total RNA. All solutions are guaranteed to be RNase-free. Invitrogen The FastTrack® 2.0 mRNA Isolation Kit has permeasured oligo(dT) cellulose powder rather than the oligo(dT) cellulose supplied in the previous kit. The procedure requires no ultracentrifugation or guanidinium lysis. The company, which markets its kits based on their recovery, reports yields of at least 40 µg of intact (as determined by agarose gel electrophoreses) mRNA from 1 x 108 Sf9 cells, with OD260/280 ratios between 1.7 and 2.2. mRNA can be isolated directly from 1x107 to 3x108 cells (depending on cell type), 0.4g to 1 g of tissue, or 0.2mg to 1 mg of total RNA in 90 minutes. The Micro-FastTrack® mRNA Isolation Kit is specifically designed for isolation of mRNA directly from samples ranging in size from 1x102 to 5x106 cells, 10-200 mg of tissue, or 100 µg of total RNA. The kit is designed for processing multiple samples simultaneously in microcentrifuge tubes, limited only by the number of positions in your microcentrifuge. Also available from Invitrogen are RNase and Protein Degrader, which is a proprietary mixture of proteases, and RNaseZAP® for removing RNase contamination from surfaces. Life Technologies Life Technologies offers MESSAGEMAKER® mRNA Isolation System. This kit contains both the TRIzol reagent, a mono-phasic solution of phenol and guanidine isothiocyanate and MESSAGEMAKER Reagent Assembly, which has a filter- syringe formatfor rapid oligo(dT) cellulose isolation of mRNA. Designed for cells or tissues, this protocol can be completed in less than three hours, yielding greater than 90 percent of mRNA from as much as 2 mg of total RNA. QIAGEN QIAGEN offers a series of Oligotex Direct mRNA Kits for purification of poly (A)+ mRNA directly from cells, tissue, or blood, and Oligotex mRNA Kits for purification of poly(A)+ mRNA from total RNA. Oligotex consists of uniformly sized, spherical latex particles that have a large surface area for dT30 oligonucleotides and hence increased binding of poly(A) sequences. The size of Oligotex particles was chosen to ensure that they remain in suspension for efficient hybridization, yet can be pelleted by brief microcentrifugation and resuspended in a small volume. The extremely small pore size of the resin excludes even low-molecular-weight contaminants. In addition, Oligotex particles can replace soluble oligo(dT) primers in cDNA synthesis reactions to yield cDNAs immobilized on Oligotex. Oligotex Direct mRNA Kits contain a guanidine thiocyanate-based buffer. Homogenized cell or tissue lysates (Oligotex Direct mRNA procedure) or total RNA preparations (Oligotex mRNA procedure) are incubated with Oligotex particles, and Oligotex/mRNA complexes are collected by a brief centrifugation step. Neither procedure requires precipitation with ethanol. QIAshredder™, a cell lysate homogenizer, is also available as an accessory for mRNA isolation. Perkin Elmer The mRNA Isolation Kit from Perkin Elmer offers a complete system for isolating mRNA directly from small amounts of starting material with the aim of providing material for PCR and other similar applications. The kit provides premeasured oligo(dT) cellulose tablets, which are combined with a lysate prepared with a non-toxic ribonuclease/protein degrader solution plus SDS. RNA is eluted from the oligo(dT) by microcentrifugation, with minimal handling. Sigma Sigma's Messenger RNA Isolation Kits can be used to isolate poly(A)+ RNA from cells or tissues in one to two hours. Using no toxic chemicals, the lysate is added directly to oligo(dT) cellulose, from which mRNA is eluted after contaminants are removed. The kit comes in two sizes, the micro kit for 20 preparations from up to 106 cultured cells or 250 mg of tissue, and the standard kit for 6 preps from up to 3 x 108 cells or 1 gram of tissue.Sigma also sells RNaseZAP for removing RNase from surfaces, and oligo(dT) cellulose separately. Stratagene Stratagene's Poly(A) Quik® mRNA Purification Kit uses push columns to quickly purify poly(A)+ RNA from total RNA. Each Poly(A) Quik push column is designed to accommodate up to 500µg of total RNA in volumes ranging from 200µl to 1µl. Poly(A) Quik push columns use applied pressure of a 10-cc Luer-Lock® syringe to force total RNA through a column of oligo(dT) cellulose. This technique eliminates the problems of slow flow rates and clogged columns. Poly(A) Quik push columns allow for the isolation of poly(A)+ RNA in less than 15 minutes. Stratagene's Messenger RNA Isolation Kits uses guanidine isothiocyanate with ß-mercaptoethanol to disrupt cells and denature proteins. mRNA isolation can be complete in less than one hour, according to the company. Worthington The Worthington mRNA Isolation Kit combines the guanidinium thiocyanate extraction and oligo(dT)-cellulose chromatography methods for the specific isolation of poly(A)+-rich mRNA from the total cellular RNA of animal, plant, and yeast cells. The kit provides all the buffers and oligo(dT) cellulose spin-columns required to isolate total and mRNA from 10 x 100 mg tissue samples or 10 x 106 cells. The protocol can be scaled up for larger amounts of tissue or higher cell concentration, and can be purchased single or as three kits together for some cost savings. Bio 101 The mRNaid Kit can be used to isolate poly(A)+ mRNA directly from a cell lysate or from a total RNA prep. Direct isolation of mRNA from a lysate is rapid, designed for use when multiple samples are needed for RT-PCR of low abundance RNA, and maximizing mRNA yields is not necessary. The kit includes a Matrix Collector Rack, which allows the Oligo(dT)30 Matrix particles that bind the RNA to be drawn onto the side of a microcentrifuge tube. This facilitates removal of unbound, non-polyadenylated RNA and washing of the matrix/mRNA complex. Biometra Oligo(dT) chains are covalently bound to the magnetic beads surface via a long chain linker to reduce steric interactions between the particle and poly(A)+ mRNA. The interaction is encouraged by the use of stringent hybridization conditions, resulting in high yields of pure, intact poly(A)+ mRNA The mRNA can be eluted in volumes as low as 30µl. All operations can be conducted at room temperature. The Magnetic Beads for mRNA should typically bind up to 10 µg of mRNA in a single application. mRNA Isolation Kit supplies buffers and reagents for 10 mRNA isolations from total RNA. Also available from Biometra are magnetic separator stands, single or multiple tube stands for use with this kit. CPG MPG® Direct mRNA Purification Kits use particle-based magnetic mRNA purification technology for the isolation of mRNA directly from tissue to provide pure, intact mRNA in 20 minutes. This method eliminates the need for initial purification of total RNA, phenol/chloroform extractions, and ethanol precipitations. The single-tube approach prevents sample loss and potential loss of rare messages. The kit contains enough reagents to isolate a minimum of 40 µg of mRNA. Increased yields can be obtained from pooling multiple rounds of mRNA isolated from the same tissue, using the same particles. The protocol is scalable. The key component of the kits, MPG Streptavidin, provides greater surface area than other magnetic particles, resulting in greater oligo(dT) binding and, therefore, generating greater mRNA capture. Typical yields of 4 µg per mg of solid phase are achieved from as little as 0.05 g of tissue (mouse liver). The messenger RNA is captured directly from tissue or cell homogenates on oligo(dT)25. The MPG Guanidine Direct mRNA Purification Kit is designed for mRNA isolation from cells and tissues with high levels of RNase. The lithium chloride containing MPG Direct mRNA Purification Kit is intended for tissues with relatively low intracellular RNase content. Cruachem Cruachem's ISOLATE mRNA Pure Prep uses a quick and simple procedure to purify mRNA from up to 100µg of total RNA. The procedure uses magnetic particles which have covalently bound oligo(dT)25 with long linker arms to prevent steric hindrance with mRNA binding. Stringent hybridization conditions result in high yields of intact, poly(A)+ mRNA suitable for many further applications. Purification is done in a single tube, which minimizes losses during handling, and mRNA is eluted in a small volume (30µl). Yields can vary with starting material, but can be expected in the range of 1 percent to 5 percent. All necessary reagents are provided, including binding, wash and elution buffers. Both single tube or multi-tube magnetic separator stands are available for purchase. Dynal With the Dynabeads mRNA DIRECT™ Kit, direct mRNA isolation is performed in 15 minutes in a single tube without any previous purification steps. The kit provides all the reagents needed for the isolation of mRNA from total RNA. Dynabeads Oligo(dT)25 are uniform superparamagnetic polymer beads, 2.8 µm in diameter with 25-nucleotide-long chains of deoxythymidylate covalently attached to the bead surface via a 5' linker group. Enzymatic downstream applications are not inhibited by the presence of the Dynabeads Oligo(dT)25. Dynabeads Oligo(dT)25 are supplied as a single reagent, or as part of ready-to-use kits. Up to 2 µg mRNA may be isolated per mg (200 µl) of Dynabeads Oligo(dT)25, depending on the tissue or cell type and the expression level of the mRNA. The protocol can be scaled up or down to suit specific sample size requirements. Miltenyi Biotec Miltenyi Biotec's mRNA Isolation Kits were developed for the purification of poly(A)+ mRNA from cultured cells, animal and plant tissue, blood or total RNA. The basis of isolation is MACS Oligo(dT) MicroBeads. Cells are lysed with a non-toxic MACS lysis/binding Buffer, mixed with the Oligo(dT) MicroBeads in solution and the magnetically labeled mRNA are purified on a MACS column. The kit contains buffers, MACS Oligo(dT) MicroBeads, and MiniMACS columns. Due to small size of MACS MicroBeads, the surface-to-volume ratio is high, resulting to efficient and rapid hybridization. The MicroBeads do not sediment, but form a stable colloidal suspension, thus, enhancing the binding of poly (A)+ mRNA to the Oligo(dT) MicroBeads. Novagen Novagen's Straight A's™ mRNA Isolation System uses Magnetight™ Oligo(dT) Particles and a set of proprietary lysis and wash buffers that can be used with animal and plant tissues, fresh or frozen, as well as cultured cells. The isolation is quick, requiring only brief centrifugation, and efficient; company data shows greater than 95 percent purity after two rounds of binding to the beads. The paramagnetic beads, which have been treated to minimize non-specific interactions, have covalently attached oligo(dT)25, and can be reused several times. The kit can be purchased with or without Separation Stand. In addition, individual components may be purchased separately. PerSeptive Biosystems BioMag mRNA Purification Kit isolates high yields of mRNA directly from cell, plant, and animal tissues in 15 minutes. BioMag Oligo(dT)20 consists of superparamagnetic particles which are covalently attached to oligo(dT)20. After base pairing between the oligo(dT) and the poly(A) tail of mature mRNA's, the particles are magnetically separated from solution, washed, and the bound mRNA is eluted from the particles in DEPC-treated water. The procedure is completed in a single tube, and does not require the use of columns, organic extractions or precipitations. Up to 18 µg of mRNA can be isolated with 1 mg of BioMag Oligo(dT)20. The kit included all the buffers and reagents necessary for 20 isolations from total RNA and 10 tissue lysate isolations (up to 1 g of total tissue). Yields are 1 mg to 5 mg per 100 µg of total RNA or from 5 x 106 cells or 2 µg to 15 µg from 50 µg to 100 µg of tissue, depending on the tissue type and quality. BioMag Oligo(dT)20 is also supplied as a single reagent. Promega Promega offers a series of PolyATtract® mRNA Isolation Systems that can be used to isolate mRNA either from total RNA or directly from cells or tissue. Based on Promega's MagneSphere® Streptavidin Paramagnetic Particles, mRNA that has been hybridized in solution to biotinylated oligo(dT) is captured using a magnetic separation stand. The PolyATtract System 1000 and PolyATtract Series 9600TM mRNA Isolation Systems allow the direct isolation of mRNA from tissue or cells, using a guanidinium based extraction buffer, while the PolyATtract mRNA Systems I through IV are designed for use with total RNA preparations. The PolyATtract Series 9600™ mRNA Isolation System, which uses a 96-well plate format, is designed for multiple, small-scale isolations of mRNA. Processing up to 96 biological samples simultaneously reduces potential sample-to-sample variation. Systems can be purchased with a magnetic stand and/or with cDNA synthesis reagents. Quantum Biotechnologies Quantum Biotechnologies' MPG® Direct mRNA Purification Kits are designed for isolating mRNA directly from cells and animal or plant tissue. The isolation takes 20 minutes and eliminates the need for total RNA isolation, oligo(dT) columns, phenol-chloroform extraction, or long centrifugations. The key component is MPG Streptavidin, which yields 4 µg per mg of solid phase. This kit comes in two versions, one with a lithium chloride based homogenization buffer and one with guanidine based homogenization, for tissues with high levels of ribonuclease. Quantum Biotechnologies also offers a kit, the MPG mRNA Purification Kit, for isolating mRNA from purified total RNA utilizing magnetic separation technology. The kit contains all the reagents necessary to isolate a minimum of 40 µg of mRNA in a single round of isolation. Roche (Boehringer Mannheim) With Roche's mRNA Capture Kit, poly (A)+ mRNA is immobilized in streptavidin-coated PCR tubes using cultured cells, tissues or total RNA preparations as starting material. The mRNA is released from cells and tissues by a lysis step that uses no hazardous organic solvents and does not require centrifugation. The poly (A)+ chain of the mRNA serves then as the hybridization site for biotin-labeled oligo(dT)20. The oligo(dT)20 has two functions: to capture (A)+ mRNA in streptavidin-coated PCR tubes and to serve as a primer for the reverse transcription reaction, which can be done after washing away the lysate supernatant. Up to 40 µg of total RNA, 5 x 105 cultured cells or 20 µg of tissue may be processed with this preparation, which takes approximately 30 minutes when starting with total RNA. Each kit provides sufficient material for capturing 192 poly (A)+ preps in PCR tubes. Vector Laboratories SCIGEN has produced magnetic particles with unique biochemistries to produce a new range of affinity purification reagents including Vector Lab's mRNA Isolation Kit. With up to 25 separate isolations per kit and 2µg to 5µg mRNA purified per mg particle, 50µg to 125µg mRNA can be isolated with one kit. The five-step protocol can be complete in 15 minutes. High capacity magnetizable cellulose oligo(dT)30 particles consist of an inert, stable solid support with low non-specific binding. All reagents are RNAse free and are prepared using sterile technique to avoid contamination. Charts The author can be reached at ldefrancesco@the-scientist.com.

Ode to Oligo(dT): Oligo(dT) Takes on a Variety of Faces in Kits for the Purification of mRNA

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