Read in this article:
Introduction
Nature and Abilities of Reverse Transcriptase
When to Apply One-Step or Two-Step RT-PCR
Choosing Between RT Priming Methods
Specific Considerations for Proofreading RT Steps
RNase H Activity
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.
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).
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.
Minimizes time required
Reduces risk of contamination
Improves sensitivity and specificity
Allows optimal reaction conditions
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.
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.
Figure 1: Overview of first strand cDNA synthesis with different types of RT primers.
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.
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.
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.
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