Getting The Message With RT-PCR

Date: July 20, 1998RT-PCR Kits Reverse transcription followed by the polymerase chain reaction (RT-PCR) has become one of the great "workhorse" techniques of today's labs. It is often used as a method for generating needed reagents, including complementary DNA (cDNA) inserts for cloning, cDNA libraries, and templates for in vitro transcription. None of the other commonly used methods for measuring the steady-state levels of individual RNAs (such as Northern or dot blotting, RNase or S1 nuclease protection assays, and in situ hybridization) come close to matching RT-PCR's sensitivity. Because of this, RT-PCR is widely used to characterize messenger RNA (mRNA) expression patterns and to compare the relative levels of mRNAs in different sample populations. When optimally performed, RT-PCR can be used to detect transcripts produced at very low levels and can identify RNAs in minute quantities of starting material. Figure 1: PCR Amplification Of cDNA Synthesized With Advantage RT-For-PCR Kits Versus Two Competitors' Kits. Varying amounts of human lung total RNA was subjected to RT-PCR using human G3PDH primers. Lanes 1-4 were done with CLONTECH's kit and 250 ng, 62.5 ng, 16 ng, and 4 ng starting material respectively; Lanes 5-8 and Lanes 9-12 were done using other kits and the same amounts of starting material. Figure provided by CLONTECH Of course, every technique has its down side. One of the major disadvantages of RT-PCR is that it is simply not as quantitative as the other methods for measuring mRNA levels. However, academic and commercial researchers alike continue to design and refine methods that use different internal or external standards to make RT-PCR as quantitative as it can be. In this article, we'll first review some basic considerations when choosing components for the reverse transcription portion of RT-PCR, then we'll highlight some of the features of currently available kits for reverse transcription and RT-PCR. In RT-PCR, an RNA template is first copied into cDNA using a reverse transcriptase (RTase), a reaction termed "first-strand synthesis." PCR is then performed to exponentially amplify the cDNA. RT-PCR protocols fall into several categories based on the number of enzymes used and whether the reverse transcription and PCR components of the procedure are performed separately. There are three basic variations of a "two-step" protocol. In some methods, the first-strand synthesis reaction is performed in one tube. Aliquots are then removed and diluted into PCR reaction mixtures in separate tubes. Many of today's "two-step" protocols use "single-tube" systems, which use buffers that are compatible with both reverse transcription and subsequent PCR. After the first-strand synthesis reaction, the tubes are opened and the DNA polymerase(s) and other PCR reagents are added. A third type of "two-step" protocol utilizes one switch-hitter enzyme to catalyze both the reverse transcriptase and PCR reactions. This variation exploits a DNA polymerase, such as Thermus thermophilus (Tth), that demonstrates reverse transcriptase activity under some conditions and DNA polymerase activity under others. A number of vendors now offer "one-step" RT-PCR systems, which reduce hands-on time and the likelihood of introducing contaminants into reaction mixtures. Most of these systems contain reverse transcriptase/DNA polymerase blends along with co-optimized reaction mixtures and enable the two procedures, reverse transcription and PCR, to be carried out successively with no additions midstream. A "one-step" kit using the single Tth enzyme is also on the market, for example. Figure 2. RAP-PCR Method. Figure provided by Stratagene. RTases from avian myeloblastosis virus (AMV-RT) and Moloney murine leukemia virus (MMLV-RT) were the first to be widely used. Many of today's RT-PCR protocols employ isolated, or more commonly, recombinant, forms of either of these two enzymes. Both of these enzymes are inactivated at elevated temperatures, but AMV-RT can withstand higher temperatures than can MMLV-RT. Recommended reaction temperatures are generally about 37ºC to 42ºC for MMLV-RT and 48ºC to 55ºC for AMV-RT. Thus, one advantage of AMV-RT is that it enables the use of higher reaction temperatures, which reduces problems associated with RNA secondary structure. On the other hand, MMLV-RT is an efficient enzyme and has a lower intrinsic RNAse H activity than does AMV-RT. The intrinsic RNA template-hydrolyzing activity of RTases can lower the yields and sizes of cDNA products. A number of currently marketed RTases are active at much higher temperatures than is AMV-RT. For example, Tth can be used to perform the first-strand synthesis reaction at temperatures up to 70°C. But be forewarned. Many of these thermostable enzymes have higher error rates than do MMLV-RT or AMV-RT. Also, remember that if you run your first-strand synthesis reaction at 70°C, you must use primers that will anneal to RNA at that temperature. (By the way, the ubiquitous random hexamer primers will not anneal to RNA templates at 70°C). Gene-specific primers designed with melting temperatures high enough to anneal at 70°C are frequently used in conjunction with these highly thermostable RTases. Many vendors now offer optimized blends of first-strand synthesis polymerases and DNA polymerases for "one-step" RT-PCR. Reaction efficiency and fidelity are two important considerations when choosing a DNA polymerase for PCR amplification. The error rates of Taq and other efficient, robustly thermostable DNA polymerases leave something to be desired. Of course, error rates depend on the enzyme and conditions, but in general, Taq and other polymerases that lack exonuclease activity make errors in the neighborhood of one per every 104 to 105 base pairs. High fidelity enzymes with "proofreading" (3' to 5' exonuclease) are much more accurate. For example, the proofreading DNA polymerase from Pyrococcus furiosus ( Pfu ) has an error rate of approximately 10-6 per base pair. However, these proofreaders can't match the yields and product sizes of Taq and the other nonproofing polymerases. Many companies now offer PCR and RT-PCR kits that feature DNA polymerase mixtures that combine a standard "workhorse" polymerase with a small amount of a proofreading enzyme. These blends provide a nice balance between fidelity and yield. For a more thorough discussion of DNA polymerases for PCR, see S. Beck, The Scientist, 12(1):16, January 5, 1998. Figure 3. Linearlity of RT-PCR Reactions. When the reaction is linear the output PCR product levels will be directly proportional to the input cDNA. Notice that although the PCR products continue to accumulate after the reaction plateaus, the rates of accumulation are no longer linear. Figure provided by Ambion. Life Technologies offers the SuperScriptTM Preamplification System for First-Strand cDNA Synthesis, which contains a genetically engineered form of MMLV-RT. Selected point mutations were introduced into the polymerase to eliminate RNase H activity while retaining full DNA polymerase activity. Life Technologies indicates that first-strand synthesis reactions catalyzed by SuperScript II RT can be carried out at temperatures as high as 50°C. Invitrogen's cDNA CycleTM Kit is an AMV-RT-based kit for the first-strand synthesis reaction. It touts a unique combination of sodium pyrophosphate, spermidine, and heat to remove secondary structure. Life Technologies' AMV Reverse Transcription System comes with oligo(dT) primers, and Roche Molecular Biochemicals and Life Science Inc. offers an AMV-RT-based kit that comes with both random and oligo(dT) primers. Roche states that AMV-RT has a high specific activity under its conditions. Promega's Reverse Transcription System uses AMV RTase and high concentrations of oligo(dT) primers to perform first-strand synthesis in 15 minutes. "Two-Step" RT-PCR Kits PE Applied Biosystems sells the GeneAmpTMRNA PCR Kit. In this kit, reverse transcription is catalyzed by MMLV-RT, and AmpliTaq® DNA Polymerase is used for PCR. The buffer system enables both reactions to take place in a single tube, but the PCR components must be added after the first-strand synthesis reaction. PE's GeneAmpTM Thermostable rTth Reverse Transcriptase RNA PCR Kit is a single-tube, single-enzyme system. First-strand synthesis is performed at 60°C to 70°C and catalyzed by rTth DNA polymerase. This enzyme acts as an RTase in the presence of manganese chloride. Perkin-Elmer points out that performing the reaction at 70°C generally circumvents the need for using RNA denaturants. After reverse transcription, a chelating buffer, magnesium chloride, and second primer for PCR amplification are added. This kit can be used to generate cDNA products of up to 1.3 kb in length. A one-step version of this kit is also available and is described in the following section. TaKaRa Shuzo Co. Ltd.'s RT-PCR products, including its RNA Amplification Kit, Ver. 2, are distributed in the United States by Panvera. This single-tube, two-step system uses AMV-RT XL (eXtra Long) for the first-strand synthesis reaction and TaKaRa TaqTM for PCR. It allows amplification of products greater than 5 kb in length. For longer products, consider TaKaRa's LA ("Long and Accurate") RNA PCR Kit, which can be used to generate products up to 12 kb. This kit uses AMV-RT XL for the first-strand synthesis and TaKaRa LA TaqTM for PCR. TaKaRa recommends its BcaBESTTMRNA PCR Kit if high levels of RNA secondary structure are a problem. The first-strand synthesis reaction can be performed at 65°C. The reverse transcription and PCR enzymes are Bca polymerase (from Bacillus caldotenax) and Bca-optimized Taq , respectively. Life Technologies offers the ThermoScriptTM RT-PCR System containing ThermoScriptTM RT, an avian RNase H-minus RTase engineered to have a higher thermal stability than AMV-RT. First-strand synthesis is performed at 50°C to 65°C. The first-strand synthesis reaction reagents are sold in kits with or without PlatinumTM Taq DNA Polymerase for subsequent PCR. PlatinumTM Taq DNA Polymerase contains a mixture of the DNA polymerase and an anti- Taq antibody. This antibody binds and inactivates the polymerase prior to the initial heat-denaturation step of PCR, thus preventing unwanted activity at inappropriate reaction temperatures. Taq is released when the antibody is denatured at high temperatures. Thus, this enzyme is referred to as giving an "automatic hot start"--that is, it does not exhibit significant activity until a certain temperature is reached and does not require wax beads or other devices for physical separation. CLONTECH offers polymerase mixes that, when used with its AdvantageTM RT-PCR kits, described earlier, allow for complete RT-PCR. Advantage mixes have been optimized for long-distance PCR and include TaqStart, which provides an antibody-mediated hot start. Figure 5 Roche Molecular Biochemicals' TitanTM One Tube RT-PCR System. Lanes 3-12: Mouse and human total RNA samples were used for the RT-PCR amplification of different transcripts ranging in size from 145 bp to 4 kb. Lanes 1 and 14: DNA Molecular Weight Marker VII. Lanes 2 and 13: DNA Molecular Weight Marker VI. Figure provided by Roche Molecular Biochemicals. Stratagene's MMLV-RT based RAP-PCR (RNA Arbitrarily Primed PCR) Kit may be of interest to those studying differential gene expression (Figure 2). During first-strand synthesis, a single 18-base arbitrary primer anneals and extends from sites contained within the mRNA. Second-strand synthesis proceeds in a similar manner during a single round of low-stringency PCR with DNA polymerase (purchased separately). PCR amplification at high stringency proceeds by virtue of having incorporated the arbitrary primer in both ends of the cDNA. A template-dependent competition determines which potential PCR products will ultimately predominate. Differences in the electrophoretic patterns of PCR products amplified from two RNA samples provide preliminary evidence for differential gene expression. Stratagene offers several different 18-base RAP-PCR primers that can be purchased with or separately from the other kit components. Ambion's QuantumRNA™ Quantitative RT-PCR module provides reagents to address questions of the abundance of transcripts among samples via relative or competitive RT-PCR. It is designed to be used in conjunction with reverse transcription reagents from Ambion's RETROscriptTM First-Strand Synthesis Kit. The QuantumRNATM relative PCR procedure involves the use of 18S ribosomal RNA as an internal standard to compensate for variations in RNA isolation, initial quantification errors, and tube-to-tube variations in RT and PCR reactions. While theoretically ribosomal RNA should be an excellent internal control for RT-PCR since its level remains essentially constant from sample to sample, in practice, its abundance is a major limitation to its utility as a control. (In order to be useful, an endogenous control must be expressed at roughly the same level as the RNA under study when amplified under the same conditions, and the reaction must be in the linear range for both the standard and test species--see Figure 3). Ambion's kit uses a novel technology, which it terms Competimers™, that allows the user to modulate the amplification efficiency of 18S rRNA, so that it can be used as an internal control for mRNAs of any abundance. "One-Step" RT-PCR Panvera Corporation distributes TaKaRa Shuzo Co. Ltd.'s One-Step RNA PCR Kit. This kit contains Reverse Transcriptase XL ("eXtra Long") and AMV Optimized Taq. There is no need to change buffers or add reagents between the reverse transcription and PCR amplification reactions. Promega's Access RT-PCR System utilizes a mixture of AMV-RT for first-strand synthesis and the thermostable Tfl DNA polymerase from Thermus flavus for PCR and can be used with oligo(dT) primers, gene-specific primers, or random hexamers. The company reports that with Access, message can be detected from as little as 7 pg of total or mRNA. U.S. distributors of DNAmp's CalypsoTM RT-PCR System include Bionexus and Tetra Link. This is a one-tube, three-enzyme system. The first-strand synthesis is catalyzed by AMV-RT. The AccuraseTM PCR enzyme blend contains nonproofreading and proofreading DNA polymerases. The nonproofreading enzyme was cloned from an Azorean hot spring Thermus isolate, whereas the proofreading polymerase was cloned from a novel deep-sea hyperthermophilic Thermococcus isolate. DNAmp indicates that the hyperthermophilic enzyme shows a high ratio of proofreading exonuclease to polymerase activity and that the CalypsoTM RT-PCR System is capable of producing good yields of long products. The AccuraseTM blend is touted to deliver a three-fold lower error rate than does Taq . Life Technologies' SuperScriptTM One-StepTM RT-PCR System is designed for performing cDNA synthesis and PCR in one tube using gene-specific primers supplied by the researcher (Figure 4). It features a blend of SuperScript II RT for cDNA synthesis and Taq for subsequent PCR. This kit does not contain RNA/primer positive controls. An RT-PCR primer and control set consisting of HeLa total RNA and b-actin primers is available separately. According to the company, the reaction can be carried out as high as 55°C. Roche Molecular Biochemicals TitanTM One-Tube RT-PCR Kit (Figure 5) amplifies fragments of up to 6 kb in length. The reverse transcriptase is AMV-RT; PCR is catalyzed by the Expand High Fidelity enzyme blend. Roche Molecular Biochemicals claims that this Taq DNA Polymerase/ Pwo DNA polymerase mix offers a three-fold increase in fidelity when compared to Taq alone. The GeneAmpTM EZ rTth RNA PCR Kit from PE Applied Biosystems contains the same enzymes as does the GeneAmpTM Thermostable rTth Reverse Transcriptase RNA PCR Kit described under the "two-step" kit section. However, this is a "one-step" kit. The RT and PCR reactions occur sequentially, with no need to add additional reagents. This kit uses a bicine buffer, which buffers both metal and hydrogen ions. It is recommended for routine screening of multiple samples in which the goal is to detect gene expression. For example, it is touted as being particularly useful in screening samples for the presence of viral sequences. This kit is not recommended for applications that require high fidelity. Epicentre's MasterAmpTM RT-PCR Kit is another "one-step/one-enzyme" kit. It uses the RetroAmpTM RT DNA Polymerase, which, like rTth Reverse Transcriptase, catalyzes both manganese ion dependent reverse transcription and magnesium ion dependent PCR amplification. To combat problems with secondary structure, Epicentre's reaction mix includes betaine (trimethyl glycine), which lowers the melting temperature of G/C rich regions. Amershan Pharmacia Biotech has Ready-To-Go RT-PCR Beads for performing single-tube RT-PCR. All the components for RT-PCR (including MMLV RT and Taq DNA polymerase) are stabilized in a "glass" matrix that is stable at room temperature when kept dry. In addition to the RT-PCR bead, the kit contains separate tubes of pd(T)12-18 pd(N)6 and positive control reaction beads. Gene-specific PCR primers can be added at the same time as cDNA synthesis primers to perform one-step RT-PCR, or PCR primers can be added after first-strand synthesis for two-step reactions. The Fine Print This article reviews a few very basic considerations for RT-PCR and summarizes a small segment of the relevant products out in the market. You will likely need RT-PCR-related products of different types, some of which may not have been covered in this article, for different experimental applications. As you research some of the kits listed in this article, you will almost certainly encounter additional products of interest. Every endeavor has a starting point, and I hope that the information presented here will serve to point you in the right direction. CPG Inc., a New Jersey-based company, developed and manufacturers a complete system for this application. CPG's SOLIDscript Solid-Phase cDNA Synthesis Kit includes the reagents for RNA isolation and the reverse transcription reaction. This kit, based on streptavidin-conjugated magnetic porous glass particles (MPG®) allows researchers to synthesize first-strand cDNA directly from poly(A)+ RNA immobilized to the beads. The entire procedure can be done in one tube in less than two hours. The result is an immobilized cDNA library that can be used as template for PCR. Differential Expression Analysis with CPG's SOLIDscriptTM Solid Phase cDNA Synthesis Kit. Figure provided by CPG, Inc. Quantum BIotechnologies in Montreal offers a kit similar to the CPG system, the QuantaScript Solid-Phase cDNA Synthesie Kit. DYNAL, one of the first companies to offer magnetic oligo(dT) beads (Dynabeads) for mRNA isolation, and holder of a patent for solid-phase cDNA synthesis using magnetic beads, does not have a system that includes reagents for first-strand cDNA synthesis. However, DYNAL has developed a protocol that details synthesis of immobilized cDNA directly from RNA captured on Dynabeads using the mRNA DIRECT Kit. Roche Molecular Biochemical's system differs slightly from the rest. Although Roche offers strepavidin coated magnetic particles, the company recommends using its mRNA Capture Kit, which contains streptavidin-coated tubes and biotinylated oligo(dT), to synthesize solid-phase cDNA for RT-PCR applications. According to Glenn Martin, the product manager for amplification and nucleic acid purification systems, Roche took advantage of its expertise in attaching enzymes and molecules to surfaces to develop streptavidin-coated tubes and other related products. Although synthesizing cDNA on streptavidin-coated tubes offers the same essential advantages as magnetic particles, Martin felt that "most people do not do one-tube RT-PCR with the magnetic beads." He felt the beads were used primarily for other applications, whereas the mRNA Capture Kit was specifically designed for "the one-step, one-tube RT-PCR customer". Martin sees the convenience of the one-tube procedure as the principle benefit of the solid-phase RT-PCR approach. He also emphasized that cDNA immobilized on the tube provides a "resource for additional RT-PCR reactions." Dr. Abbie Esterman, the developing scientist of the SOLIDscript system at CPG, agrees with Martin and mentioned that "since it [the immobilized cDNA library] can be stored in the at 4°C and only a small amount of the library is needed for each RT-PCR reaction, you have a ready-to-use template for your gene whenever you need it." Dr. Swayampakula Ramakanth, a scientist at Epoch Pharmaceuticals, Inc that uses the CPG SOLIDscript kit, agrees that the principle advantage of this approach is convenience. He uses SOLIDscript because "it is fast" and he "gets enough cDNA to run RT-PCR reactions within a few hours." He did believe that more standard approaches might yield greater amounts of cDNA; however, he noted, "The yield is fine for PCR." Using Roche Molecular Biochemical's system, Dr. Gilbert Cote, Assistant Professor of Medicine at University of Texas, MD Anderson Cancer Center, also emphasized convenience, mentioning that the system provides "great reproducibility and is a great timesaver." Cote added, "It takes half the time of the standard RNA isolation and purification procedure we used to use." When asked about the yield, Dr. Cote commented that "there is no way to know what the yield is because you base the prep on a set number of cells and never quantitate the RNA." Although the one-tube approach is quick and easy, Dr. Cote mentioned that doing the reverse transcriptase reaction in the same tube that contains all the RNA effectively means that you have used up your entire RNA sample in one reaction. You can go back and perform PCR from the cDNA at any time, however, you need to collect new RNA to do any other prodecure. In addition to convenience, data generated during development of the CPG SOLIDscript system indicates that the immobilized solid-phase cDNA library may increase message representation. According to Dr. Esterman, rare messages, which might be lost by an elution procedure, are maintained since the cDNA synthesis is done right on the magnetic beads. Paul Diehl is a free lance writer based in Perth Amboy, N.J. He can be reached at paul_vfocus@msn.com Deborah Wilkinson holds a doctorate in biochemistry and currently works as a freelance writer in Memphis, Tenn. She can be reached at deborahwilkinson@sprynet.com RT-PCR Kits

Getting The Message With RT-PCR

Interested in reading more?

Become a Member of