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Choosing the correct enzyme and reaction set-up for reverse transcription and cDNA synthesis is critical for obtaining high yields of quality full-length cDNA with full gene representation. Roche offers a comprehensive selection of reverse transcription products, including Transcriptor product family and LightCycler® Kits:
- Benefit from enzymes with consistent performance across a broad dynamic range.
- Increase temperature to transcribe RNA with secondary structures.
- Use proofreading activity for demanding applications.
- Reduce pipetting steps by using a convenient cDNA master.
Select the Optimal Enzyme for RT-PCR
A major factor to consider in reverse transcription polymerase chain reaction (RT-PCR) is the choice of the appropriate reverse transcriptase. Roche has developed and optimized a wide variety of different enzymes and enzyme blends. Each of these products has different enzymatic properties and one may be more suitable for a specific experiment than the other. The information given below outlines the individual properties of each enzyme and may help you determine the optimal enzyme or enzyme blend for your specific application.
|Tth DNA Polymerase||Titan One Tube RT-PCR System||Titan One Tube RT-PCR Kit|
|Composition||Thermostable DNA polymerase with intrinsic reverse transcriptase activity.||AMV reverse transcriptase and Expand High Fidelity Enzyme Blend.|
|Product Size||Up to 1 kb||Up to 6 kb|
|Reaction Temperature||+55 to +70°C||+45 to +60°C|
|RNase H Activity||-||++|
|Sensitivity - Total RNA||+||1 pg to 1 µg|
|Sensitivity - mRNA||+||++|
|Sensitivity - Viral RNA||+||+|
|Sensitivity - RNA Rich in Secondary Structures||+||+|
|Fidelity||Highly processive 5' - 3' polymerase, lacking 3' - 5' exonuclease or proofreading activity.||+|
|Incorporation of Modified Nucleotides||Yes||Yes|
|Buffer System||Both a PCR and RT-PCR buffer are included.||A single reaction buffer is included.|
Two-step RT-PCR offers maximum flexibility with respect to primer choice, enzyme, temperature and multiple transcripts analysis
|Reverse Transcriptase M-MuLV||Expand Reverse Transcriptase||Reverse Transcriptase AMV||First Strand cDNA Synthesis Kit||cDNA Synthesis System|
|Product Size||10 kb||14 kb||12 kb|
|Priming||Specific Oligo(dT) Random hexamer||Specific Oligo(dT) Random hexamer||Specific Oligo(dT) Random hexamer|
|Reaction Temperature||+37°C||+42°C (up to +50°C)||+42°C (up to +60°C)|
|RNase H Activity||+||-||++|
|Sensitivity - Total RNA||+||++||+|
|Sensitivity - mRNA||+||++||++|
|Sensitivity - Viral RNA||+||+||+|
|Sensitivity - RNA Rich in Secondary Structures||+(+)||+||+|
|Incorporation of Modified Nucleotides||Yes||Yes||Yes|
|Carryover Prevention||Not determined||Not determined||Not determined|
|Buffer System||A single reaction buffer is included. Separate vials of DMSO, DTT, and Betain for optimization of reaction conditions are also included.||A single reaction buffer is included. A separate vial of DTT for optimization of reaction conditions is also included.||All necessary reagents are included.|
Tips for Nucleic Acid Handling
> Precautions for Handling RNA - Nucleic Acid Purification
Read about how to produce an RNase-free environment.
> DNA-Free RNA Isolation
Read about how to detect and eliminate genomic DNA.
> Reverse Transcription
Read about the optimal priming method and how to optimize the RT reaction.
> Cloning and Gene Synthesis - Protein Expression
Read about how to minimize errors, choose the best tag, influence protein yield, and more.
Reverse transcription is the process by which a reverse transcriptase enzyme mediates the creation of a DNA complement (complementary DNA or cDNA) from an RNA strand. The discovery and use of reverse transcriptases has greatly improved knowledge in the area of molecular biology. Reverse transcriptases are used for gene expression analysis, to create cDNA libraries from mRNA and, along with other enzymes, allow cloning, sequencing, and characterization of RNA.
In 1987, Powell et al. described a technique that extended the power of PCR to the amplification of RNA. This technique, RT-PCR, uses a reverse transcriptase to convert RNA into cDNA, followed by a thermostable DNA polymerase to amplify the cDNA to detectable levels, thus making it possible to use PCR to detect and analyze mRNA transcripts and other RNAs present in low abundance.
Nature and Abilities of Reverse Transcriptase
Reverse transcriptases first discovered in 1970 by Howard Temin and David Baltimore, can be found in reverse transcribing viruses, such as the human immunodeficiency virus (HIV) and the hepatitis B virus. The most commonly used reverse transcriptases are the AMV reverse transcriptase from the avian myeloblastosis virus and the M-MuLV reverse transcriptase from the Moloney murine leukemia virus.
The reverse transcriptase enzyme is encoded and used by reverse transcribing viruses, which use the enzyme during the process of replication. In vitro, the process is priming dependent and functions at a temperature of +40 to +50°C, depending on the properties of the reverse transcriptase used. However, the process of reverse transcription is extremely error-prone, because the reverse transcriptase (unlike any other DNA polymerases) has no proofreading ability due to its viral origin.
Native reverse transcriptases are multifunctional enzymes. Their DNA polymerase activity allows the transcription of ssRNA and ssDNA. In addition, the endogenous RNase H activity in some reverse transcriptases leads to the degradation of the RNA from an RNA:DNA hybrid. The degradation of the original RNA is a crucial issue for the quality of the cDNA in the subsequent PCR step (see below).
When to Apply One-Step or Two-Step RT-PCR
The PCR technique can be applied only to DNA strands. However, with the help of reverse transcriptase, RNA can be transcribed into DNA, thus making PCR analysis of RNA molecules possible. There are two strategies that combine reverse transcription and PCR: one-step RT-PCR and two-step RT-PCR. If the RT step is performed in the same tube with PCR, the process is called one-step PCR. When the PCR and RT are performed in separate tubes, the process is called two-step PCR. With either method, one can usually obtain sufficient cDNA from 1 µg total RNA.
Advantages of One-Step PCR
Advantages of Two-Step PCR
Minimizes time required
Allows optimal reaction conditions
Reduces risk of contamination
Improves sensitivity and specificity
Provides maximum flexibility
For two-step RT-PCR, to avoid the addition of a purification step between the RT and the PCR reactions, the buffer used for the RT reaction must also be compatible with the subsequent PCR step.
Choosing Between RT Priming Methods
There are three choices of RT primer: oligo(dT), random primers, or a gene-specific primer. The priming strategy has a strong impact on the workflow applied, since each priming method has prerequisites and consequences.
Oligo(dT) priming is used to receive full-length copies of the mRNA. For cDNA library construction or cDNA labeling applications, oligo(dT) primers are almost always used to prime cDNA synthesis. Anchored oligo(dT)18 primers are designed to bind at the beginning of the poly(A) tail (rather than randomly within the often long tail) to generate full-length cDNAs. This avoids mispriming and unnecessary reverse transcription of the long poly(A) tail. Since the 5´ ends of long mRNAs are often underrepresented in cDNA mixtures, this primer is preferred for most applications.
However, if the message is long or does not have a poly(A) tail (as with prokaryotic mRNA), then random hexamer primers are used. Random hexamer primers bind throughout the entire length of RNA, ensuring reverse transcription of all RNA sequences due to their random structure.
A mixture of both random hexamer and oligo(dT) is possible, as well.
The third choice is a gene-specific primer. Gene-specific primers enhance sensitivity by directing all of the RT activity to a specific message instead of transcribing everything in the mix. If you are performing a one-step RT-PCR, gene-specific primers are used because the RT primer is also your reverse primer for the PCR step.
Panel A: Oligo(dT)n primer
(in this case n = 18)
Panel B: Anchored oligo(dT)n primer
(in this case n = 18). Reverse transcription starts at the very beginning of the poly(A) tail.
Panel C: Sequence-specific (gene-specific) primer.
Panel D: Random hexamers.
V = A, C, or G
B = C, G, or T
N = A, C, G, or T
Note: Use random primers at a final concentration of 60 µM for an optimal reaction result.
Figure 1: Overview of first strand cDNA synthesis with different types of RT primers.
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Specific Considerations for Proofreading RT Steps
Retroviral reverse transcriptases commonly used for cDNA synthesis exhibit a higher error rate than other DNA polymerases used in nucleic acid analysis techniques. This lack in accuracy leads to a significant number of base exchanges or frameshifts, which are further propagated in subsequent PCR reactions. High fidelity (proofreading) PCR enzymes have been available for many years; Roche offers a high accuracy reverse transcriptase, Transcriptor High Fidelity cDNA Synthesis Kit, which is further able to synthesize high yields of full-length cDNA.
High mutation rate is a hallmark of retrovirus replication. This originates in the mechanism of genome replication by the viral-encoded reverse transcriptase, which converts the genomic RNA of the virus to double-stranded DNA (dsDNA). During this process, reverse transcription produces frequent replication errors. One accepted explanation of this inaccuracy is the lack of RT 3´-5´ exonuclease activity. The naturally high error rate of reverse transcriptases is not optimal for many applications.
The core component of the Roche kit is the Transcriptor High Fidelity Reverse Transcriptase, a blend of a recombinant reverse transcriptase and a proofreading mediating enzyme. The synergy between both enzymes is the key to the enzyme blend's ability to reverse transcribe RNA templates with 7-fold higher fidelity compared to other commonly used reverse transcriptases.
RNase H Activity
If the RNA template is not degraded after first strand cDNA synthesis, it can bind to the newly synthesized cDNA and restrict the accessibility of primers during subsequent PCR amplification. RNase H-mediated destruction of the template can prevent this problem and improve the sensitivity of RT-PCR analysis. However, an additional RNase H incubation step prolongs the reaction time and incurs additional costs for the often expensive RNase H. The Roche Transcriptor Reverse Transcriptase has endogenous RNase H activity ideal for digesting the original RNA template.
Polumuri et al. (2002 BioTechniques 32, 1224-1225) showed that reverse transcriptases without RNase H activity can limit the sensitivity of RT-PCR detection. Polumuri and colleagues tested the effects of RNase H treatment on RT-PCR detection sensitivity using SuperScript II M-MuLV RNase H- RT to amplify 3 genes (NCX1, NCX2, NCX3). Of these 3 targets, one (NCX2) was detected much more readily when an RNase H step was included after the reverse transcription.
Related Products for Conventional RT-PCR
> RNase Inhibitor
> DNases and RNases
> Cell Lysis Kits
> Uracil-DNA Glycosylase
> 5'/3' RACE Kit, 2nd Generation
Protector RNase Inhibitor
Recombinant from rat lung, special quality for Molecular Biology.
Inactivate a wide spectrum of RNases, including RNase A, RNase B, and RNase T2. Protect mRNA and total RNA during isolation and cDNA synthesis. Application Protector RNase Inhibitor is used to protect mRNA in cDNA synthesis reactions, RT-PCR, including quantitative RT-PCR (e.g., with the LightCycler® Instruments), in vitro transcription/translation systems, RNase protection assays, in vitro RNA synthesis, and in vitro virus replication. It is also used to prepare RNase-free antibodies. It is useful in any application where RNases could be a potential problem.
Rely on a thermostable RNase inhibitor.
Use Protector RNase Inhibitor alongside thermostable reverse transcriptases like Transcriptor Reverse Transcriptase.
Benefit from a wide range of reaction conditions.
Protector RNase Inhibitor is active at pH 5.0 to 9.0 and at temperatures between +25° to +55°C (partial activity is still measurable at +60°C).
Eliminate interference in different RNA analysis applications.
Maintain performance even when adding 16 times the standard concentration.
Insist on a highly purified preparation.
Each batch is function tested using techniques like quantitative RT-PCR to ensure the absence of endonucleases, ribonucleases, or nicking activity.
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DNase I recombinant, RNase-free
from bovine pancreas, expressed in Pichia pastoris.
|Endonuclease for double- and single-stranded DNA, free of ribonuclease and protease, according to the strict current quality control.||Isolation of DNA-free RNA.||Ideal for the isolation of RNA, since it is free of RNases, according to the strict current quality control. Recombinant enzyme, free of animal components.|
from bovine pancreas
|RNase mixture, free of contaminating DNases, according to the strict current quality control.||Isolation of RNA-free DNA.||Ideal for isolation of DNA, since it is free of DNases, according to the strict current quality control. Unlike most RNase preparations, does not need to be boiled prior to use.|
from bovine pancreas
|Pyrimidine-specific endonuclease that acts on single-stranded RNA.||Isolation of genomic DNA.||Use in many DNA isolation procedures.|
|RNase H||Endonuclease that cleaves RNA in RNA:DNA hybrids.||Elimination of RNA template after first strand cDNA synthesis.||Eliminates potential source of PCR errors. Increase accessibility of primers during subsequent PCR.|
from bovine pancreas
|Crude mixture of RNases.||Isolation of genomic DNA.||Use in many DNA isolation procedures.|
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|RNA, MS2||From bacteriophage MS2||To stabilize RNA during cDNA synthesis. For studies which use natural RNA in an in vivo and in vitro protein-synthesizing system; also used in initiation, elongation, and termination of polypeptide synthesis.||Increase RT-PCR performance|
|RNA||Total RNA from Saccharomyces cerevisisae||For studies which use natural RNA in an in vivo and in vitro protein-synthesizing system. The product is used as a carrier RNA for in situ hybridization.||Use as carrier RNA.|
|RNA, 16S- and 23S-ribosomal||From E. coli MRE600||Use this RNA as molecular weight standard in gel electrophoresis and ultracentrifugation, and for studies of the structure and function of ribosomes.||Defined length of 1,500 and 2,900 nucleotides.|
|Poly(A)||Polyadenylic acid||RNA carrier for precipitation||Readily soluble salt|
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Cell Lysis Kits
RealTime ready Cell Lysis Kit
The RealTime ready Cell Lysis Kit includes an advanced Lysis Buffer that creates cell lysates within 5 minutes at room temperature. Prior to the lysis step, Protector RNase Inhibitor must be added to the Lysis Buffer to prevent RNA degradation during both cell lysis and storage. The cell lysates can be conveniently stored at -15°C to -25°C for several months, or stored at +2°C to +8°C for a maximum of 2 days, if not immediately processed for the two-step real-time RT-PCR experiment. DNase treatment can be conveniently and time effectively integrated into the cDNA synthesis step. In combination with fast cDNA synthesis and fast real-time PCR protocols, gene expression experiments using cultured cells can be conducted in less than 1,5 hours.
The RealTime ready Cell Lysis Kit is designed for rapid, high-performance gene expression studies using cultured cells in two-step, real-time RT-PCR applications. The kit uses an advanced one-step protocol for lysing 3-30,000 cells within 5 minutes at room temperature. The resulting lysate can be used directly in cDNA synthesis, eliminating the time-consuming RNA purification step. Finally, the cDNA can be analyzed in real-time PCR experiments at any throughput.
Due to the user-friendly packaging and easy-to-use protocol, the RealTime ready Cell Lysis Kit can be easily adapted to all experimental and throughput requirements. In addition, the kit easily integrates into all manual and automated cell-based gene expression workflows.
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|Uracil-DNA Glycosylase, heat-labile||Recombinant from marine bacterium BMTU 3346. This enzyme shows lower thermostability and is therefore, easier to inactivate.||U-DNA cleavage can be used to increase the efficiency of site-directed mutagenesis. For this purpose, uracil is incorporated in vivo into the target DNA. After primer annealing, enzymatic filling-in, and ligation, the selection for the mutated strand is performed by enzymatic degradation of the original non-mutated uracil (which contains the wild-type strand) using UNG. A further application is carryover prevention for PCR. During PCR, dUTP can be incorporated into the PCR product. Before a subsequent amplification reaction, contaminating PCR products are degraded using Uracil-DNA Glycosylase. The enzyme is inactivated at the beginning of the amplification reaction by heating to +95°C. Simultaneous strand cleavage of the U-DNA is achieved during this heating step.||
|LightCycler® Uracil-DNA Glycosylase||
||LightCycler® Uracil-DNA Glycosylase (LightCycler® UNG) is ideally suited for preventing carryover contamination between PCRs. This technique involves incorporating deoxyuridine triphosphate (dUTP) into all amplification reactions, and pretreating all successive LightCycler® FastStart enzyme-based PCR mixtures with LightCycler® UNG.||LightCycler® UNG is designed only for use with the FastStart enzyme or FastStart enzyme-based LightCycler® Kits.|
|Uracil-DNA Glycosylase||Recombinant from E. coli K 12.||U-DNA cleavage can be used to increase the efficiency of site-directed mutagenesis. For this purpose, uracil is incorporated in vivo into the target DNA. After primer annealing, enzymatic filling-in, and ligation, the selection for the mutated strand is performed by enzymatic degradation of the original non-mutated uracil (which contains the wild-type strand) using UNG. A further application is carryover prevention for PCR. During PCR, dUTP can be incorporated into the PCR product. Before a subsequent amplification reaction, contaminating PCR products are degraded using Uracil-DNA Glycosylase. The enzyme is inactivated at the beginning of the amplification reaction by heating to +95°C. Simultaneous strand cleavage of the U-DNA is achieved during this heating step.||
Amplify DNA sequences from a messenger RNA (mRNA) template between a defined internal site and unknown sequences of either the 3' or 5' end of mRNA. The kit contains all necessary components for performing RACE, including Reverse Transcriptase, dNTPs, buffers, controls, primers, and the High Pure PCR Product Purification Kit.
- Robust performance: Recombinant Transcriptor Reverse Transcriptase allows procession through regions of difficult secondary RNA structure.
- Convenient: Function and expression studies of either 5' or 3' end of the RNA can be performed with the same kit.
- Reliable: dA tailing of cDNA with Recombinant Terminal Transferase decreases the likelihood of inappropriate truncation.
- Reproducible: Oligo dT-anchor primer with non 3'dT ensures correct binding to the inner end of the poly (A) tail.
- Produce long fragments: Generate cDNA up to 14 kb in length with Transcriptor Reverse Transcriptase.