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Reverse Transcription

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Overview

Reverse transcription is a process that involves a reverse transcriptase (RTase), an enzyme that uses RNA as the template to make complementary DNA (cDNA). This process is the exact opposite of the naturally occurring DNA transcription in which RNA is synthesized using DNA as the template (1). RTases are typically found in retroviruses where they aid in the replication and incorporation of these RNA viruses into host genomes (1)(2)(3).

RTase includes 2 crucial enzymatic components – a DNA polymerase that can operate on both RNA (first strand synthesis) and DNA (second strand synthesis), and a ribonuclease H (RNase H) domain (Figure 1)(1). Each of these domains contributes to the process of cDNA synthesis from RNA. RTase was first isolated from Murine Leukemia Viruses (R-MLV) and Rous Sarcoma Viruses (RSV), and since then, there are various applications in molecular biology that involve reverse transcription (1). For example, reverse transcription-polymerase chain reaction (RT-PCR) can be used to detect if certain genes are being expressed, which can assist in diagnosing genetic diseases (4) and detecting cancers (5). With RT-PCR technology, RNA that is fragmented, degraded, or even in small amounts can be used for downstream applications (6).

RT Domains


Figure 1 – The reverse transcriptase domains are shown here. The polymerase domain includes the RNA and DNA-dependent domains. The nuclease domain is the RNase H domain. The blue and green area is either a DNA/RNA hybrid or DNA/DNA helix threading through the polymerase.


Theory

Reverse transcription begins by converting the desired RNA transcript into cDNA with using the reverse transcriptase's (RTase) RNA-dependent DNA polymerase (7). RTases have a very low accuracy during synthesis due to its lack of proofreading ability, which is why viruses that use RTases create many mutations (8).

To start off the process, part of a transfer RNA (tRNA) is unwound and is used as the primer that binds to the primer binding site (PBS). The cDNA that is first created is single stranded, and the production of this strand is therefore called first strand synthesis or minus strand synthesis (1). The RNA-dependent DNA polymerase uses the RNA genome as a template to synthesize the cDNA. Second strand synthesis, or plus strand synthesis, is the creation of the second strand of DNA, which uses the first strand of DNA as a template. This uses the DNA-dependent DNA polymerase domain of the RTase.

When the RTase is transcribing RNA into cDNA, its structure is shaped in a specific way such that its RNase H domain can cleave the RNA strand shortly after that part has been synthesized into cDNA. RNase H cleavage is necessary so that strand transfer or “jumps” can occur throughout the process. A strand transfer reaction is when a part of the newly synthesized DNA translocates to another part of the template strand it is using after RNase H has cleaved the RNA it is annealed to.

The RNA template contains regions called the R region and U5 region at the 5’ end, and another R region and U3 region at the 3’ end. After reverse transcription takes place, there is a U3, R and U5 region at either end of the DNA transcript. These regions are called long terminal repeats (LTR) and this is why the DNA transcript is always slightly longer than the original RNA it was created from (Figure 2). The LTRs are created when the strand-transfer reactions transpire (8).


RNA template


Figure 2 – The RNA template (viral RNA) contains an identical R region on either end. There is a 5’ U5 region and a 3’ U3 region before reverse transcription takes place. The PBS is near the 5’ end on the template RNA and the PPT is closer to the 3’ end. After reverse transcription, the DNA contains two identical long terminal repeats (LTRs) on either ends, making it longer than the RNA.


The detailed steps of Reverse Transcription are as follows (1) (8) (Figure 3):

  • First strand synthesis starts with the tRNA acting as a primer and partially binding to the PBS on the RNA template 5’ end (the entire tRNA is not bound to the template).
  • The tRNA extends from its 5’ to 3’ end (this is anti-parallel to the RNA template) using the RNA with the PBS as a template.
  • It is extended through the U5 and R region on the template RNA until it reaches the end of the RNA transcript. This creates a short strand of DNA that is termed a minus strand strong-stop DNA ((-)sssDNA).
  • RNase H cleaves the template RNA so that the newly synthesized cDNA is not annealing to anything, which is when the first strand transfer/jump occurs.
  • The (-)sssDNA R region anneals to the complementary R region on the 3’ end of the template RNA.
  • First strand synthesis can now continue. (-)sssDNA is extended along the entire length of the template until the PBS on the 5’ end of the RNA genome.
  • RNase H continues to degrade the template during this synthesis. However, the RNA template has a part called the polypurine tract (PPT) region within the sequence that is more resistant to the effects of RNase H so it is not degraded and actually acts as the primer for second strand synthesis.
  • Second strand synthesis now begins from the PPT region. The second strand of DNA extends in the opposite direction as the first strand of DNA. Once it copies the U5 end region on the first strand, it creates a short portion of DNA called the plus strand strong-stop DNA ((+)sssDNA).
  • The RNase H causes the tRNA primer on the first strand to fall off so that the PBS on the second strand can now bind to the PBS on the first strand.
  • Each strand is now simultaneously a template for one another. They are both extended in opposite directions - the second strand still having to synthesize the majority of its strand while the first strand only needing to synthesize the rest of its 3’ LTR region.
Reverse transcription process


Figure 3 – The reverse transcription process in viruses. A) tRNA (green) binds to the PBS. B) First strand (red) extends until the end of the RNA template (black). RNase H digests the template. C) (-)sssDNA “jumps” to the 3’ end of RNA template. D) First strand synthesis continues extending towards the 5’ end of the RNA template. RNase H degrades the template except for the PPT region. E) The entire first strand is completed and the complementary RNA is degraded. Second strand synthesis begins (blue) using the RNase H resistant PPT region as a primer. The first strand is used as a template for the second strand. F) RNase H causes the tRNA to fall off once the (+)sssDNA has been synthesized. G) The second strand transfer reaction occurs in which both PBSs on the first and second strand anneal to one another. H) Both DNA strands use one another as a template for DNA synthesis. There is no longer any RNA left to degrade. The final product is presented here.


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RT-PCR

RT-PCR is used to detect gene expression by amplification of the RNA molecule through either end-point or real-time PCR (9). Reverse transcription begins by reverse transcribing the desired mRNA transcript into cDNA with the RTase (7). The cDNA that is first created is single stranded and is sequentially used as the template for PCR (Figure 4) (7). The RT-PCR technique is very useful because of its high sensitivity to low input RNA quantities (9). There are various types of primers that can be used for this process - oligo(dT) primers, random primers or gene specific primers (Figure 5). The primers that are used may target a known mRNA transcript that will lead to the detection of a desired transcript or may simply transcribe the entire RNA profile (7).


Types of Reverse Transcriptases

TaqMan probes are hydrolysis probes that utilize the 5’ to 3’ exonuclease activity of DNA polymerase. They are dual labeled with a fluorescent dye (fluorophore) on the 5’ end and a quencher dye on the 3’ end. Several different types of reporter dyes can be used in TaqMan probes, including FAM, TET, or JOE, and different types of quenchers can be used as well, including TAMRA, BHQ and MGB (13)(14). Choosing different reporters will depend on the instrumentation used for the experiment. Also, if different reporter dyes are used in multiplex experiments to detect multiple oligonucleotide sequences, the emission maxima (the peak of emission spectrum) of reporter dyes must have a difference of at least 15 nm. When choosing the quencher dye that pairs with the reporter, one must consider both FRET and static quenching. Since FRET works by transferring energy from the reporter to the quencher, it is essential for the emission spectrum of the reporter to overlap with the absorbance spectrum of the quencher. In static quenching, the structure of the dyes is crucial when pairing the quencher and the reporter (13)(14)(15).


A primer binds to the RNA and reverse transcribes it into cDNA.


Figure 4 – A primer binds to the RNA and reverse transcribes it into cDNA. RNA is simultaneously degraded after that part of the template has been used. The single stranded cDNA is then used as a template for PCR.


The two RTases that are frequently used in molecular biology are the Avian Myeloblastosis Virus Reverse Transcriptase (AMV-RT) and Moloney Murine Leukaemia Virus Reverse Transcriptase (MMLV-RT). When deciding on the materials being used for the reverse transcription reaction in RT-PCR, the secondary structure of the RNA transcript should be considered as it can cause difficulties in the process (10). AMV-RT is more stable than MMLV-RT as it can function under temperatures up to 55°C and can assist in preventing errors that arise from the secondary structures (10). The RNase H activity in MMLV-RT is much lower than AMV-RT so the RNA may not be degraded as readily. Regardless, any amount of RNase H can still cause problems for DNA synthesis in vitro (10). RNase H inhibitor can be added to increase the yield and size of the cDNA. Another aspect to note is that MMLV-RT can create products up to 7 kb and use a variety of templates (ssDNA, SSRNA, or RNA-DNA hyrbrid) whereas AMV-RT can create an even longer products of 12.3 kb and can use any type of primer - oligo(dT), random, or gene-specific (11) (12). The benefits and downfalls of each should be taken in consideration when deciding which to use.


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Types of Primers

There are a variety of primers that can be used for reverse transcription depending on the experimental goals. Gene-specific primers are the most efficient primers, next are oligo(dT) primers and thirdly random primers are the least efficient (typically random hexamers are used). However, even though gene-specific primers (Figure 5a) are the most efficient, they are not always desirable because only the target gene is amplified, thereby preventing an internal control of being co-amplified (14). The second type of primers, oligo(dT) primers (Figure 5b), are very useful when the entire messenger RNA (mRNA) transcript is desired (18). However, if the RNA is fragmented or if the secondary structure is too complex, cDNA synthesis for that fragment may not occur. These types of primers anneal to the 3’ end poly(A) tail and begins transcription from there. Lastly, random primers (Figure 5c) are useful when a large abundance of cDNA must be synthesized. This type of primer can work around the problems of secondary structures and can even use degraded RNA as the starting material (18). However, it has also been shown to display an overestimate of the total number of RNA (14). Each of these primers is specifically chosen to tailor to the exact procedure.


Sequence/gene-specific primers bind to an intended site so that the desired transcript can be converted into cDNA.


Figure 5 – a) Sequence/gene-specific primers bind to an intended site so that the desired transcript can be converted into cDNA. b) Oligo(dT) primers bind to the template such that the entire template RNA is transcribed into cDNA c) Random primers bind throughout the entire RNA template. This is why secondary structures do not pose as much as of a problem as with other primers.


Downstream applications

Once reverse transcription is completed, the resulting cDNA can be quantified through various methods, such as end-point PCR or real-time PCR (15).


End-point PCR

Quantification of end-point PCR typically includes the use of fluorescent dyes (ie. Ethidium bromide), P32 phospholabelling, or by scintillation counting (16). Depending on the experimental objective, there are three different approaches to end point RT-PCR:

  • Relative,
  • competitive,
  • and comparative

Relative end point RT-PCR involves an internal control that is amplified at the same time as the target RNA (10). The internal control does not create competition for the target RNA in the reverse transcription process because it lacks similar primer binding sites or internal sequences. The linear range of the control and target are later compared such that it can be used for normalization. In other words, by using an internal control, one can account for amplification rates and other variations in the process (10) (16).


In contrast, competitive end point RT-PCR involves the use of a synthetic RNA that contains similar primer binding sites and internal sequences, often only a slight variation, such that it becomes a competitor to the target RNA for the amplification reagents. Competitor RNA are added to numerous PCR tubes in varying amounts and the amount of PCR product is later correlated to the known concentration. The abundance of the target RNA can then be calculated based on the correlation and this gives absolute quantification of the target RNA (11)(16).


The third approach, comparative RT-PCR, is the least complicated method of the three end-point RT-PCR methods. In this method, two different RNA transcripts compete with one another for amplification reagents and can later be compared in abundance using an external standard curve (16).


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Real-time PCR (qPCR)

Real-time PCR is another commonly used technique after a reverse transcription reaction. The main advantage of using this method is that it shows DNA amplification in real-time by detecting fluorescence in DNA probes. There are various fluorescence probes, for instance, SYBR green (binds to double stranded DNA), Taqman probe, or molecular beacons (including scorpion probes) (17). Please read our Knowledge Base on Real-time PCR (qPCR) for more information.


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One-step and two–step RT-PCR

There are two types of RT-PCR: one-step and two-step (Figure 6). For one-step, the reverse transcription reaction and PCR reaction occur in a single tube, whereas in two-step RT-PCR the two reactions occur in two separate tubes (13).

Each of these methods has their own advantages and disadvantages. One-step is reported to limit experimental variation because it does not require the transfer of reaction contents to another tube. The one-step method requires the use of gene-specific primers therefore it is more suitable when a known gene is being targeted for study. One of the main advantages of one-step is that it is less expensive and less time consuming because fewer steps are required (14).

However, the two-step method is generally more accurate because of the choice to use random, oligo (dT) primers, or sometimes even gene-specific primers (14). Depending on the choice, one can allow for a greater range of RNA to be converted into cDNA. In addition, an advantage of using two tubes creates an opportunity to remove the primers after the reverse transcription reaction thereby decreasing the amount of primer dimers present in the subsequent PCR. Since RNA is unstable, the two-step method also offers a choice to store the more stable cDNA separately. By doing so, it will allow a series of subsequent PCR reactions to use the same pool of cDNA originally created. The downside of the two-step method is that since the samples are being moved to another environment (tube) there is a greater risk of contamination (13). The protocols for both of these approaches are very similar except for the number of tubes and primers used.


Differences between one-step and two-step RT-PCR


Figure 6 – A simplified flowchart is shown here highlighting the differences between one-step and two-step RT-PCR.


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Experimental Notes

The following is generally what needs to be included for an end point RT-PCR (it is subject to change depending on the specific protocol).


Reverse Transcription:

  • RNA transcript
  • Choice of primers: oligo(dT), gene-specific or random
  • 100 mM each of dNTPS (dATP, dTTP, dCTP, dGTP)
  • Reverse transcriptase
  • Reverse transcription buffer (details included below)
  • 100 mM each of dNTPS (dATP, dTTP, dCTP, dGTP)
  • DEPC treated or nuclease free water
  • RNase inhibitor (optional – RNAse H can interfere with synthesis (10))

PCR:

  • DNA Template
  • Taq DNA polymerase
  • DEPC treated or nuclease free water
  • 100 mM each of dNTPS (dATP, dTTP, dCTP, dGTP)
  • Forward and reverse primers

Materials for performing gel electrophoresis after PCR is completed:

  • Agarose
  • TE buffer
  • Loading dye
  • Ethidium Bromide

RT Buffer composition:

  • 50 mM Tris-HCl (pH 8.3)
  • 250 mM KCl
  • 5 mM MgCl2
  • 10 mM DTT (depending on exact protocol, this may not be included)
A basic outline of the steps before, during, and after RT-PCR


Figure 7 – A basic outline of the steps before, during, and after RT-PCR.


Sample Protocol:(Figure 7)

Two-step RT-PCR (7)


  • 2 µg RNA
  • RNA must be denatured - heat 2 µg of RNA at 65˚C for 5 minutes
  • Put denatured RNA on ice and setup tube for reverse transcription

    • 2 µg RNA
    • 20 µl RT buffer
    • 2.5 µl dNTP mix
    • 2.5 µM Primer (random, oligo(dT), or gene-specific)
    • 2.5 U Reverse transcriptase
    • Remaining amount to make a total 50 µl reaction - DEPC treated or nuclease free wate
  • Put in a thermocycler for 1 hour at 37-42˚C (the temperature may vary depending on the RTase used)
  • Denature the single stranded DNA by incubating the tube at 95˚C for 2 minutes. Place on ice
  • Setup PCR reaction

    • 2.5-10 µl RT reaction product
    • 5 µl 10X PCR buffer
    • 1 µl Forward primer
    • 1 µl Reverse primer
    • 2.5 µl dNTP mix
    • 0.5 µl Taq DNA polymerase
    • Top reaction up to 50 µl with PCR water
  • Run in thermocycler as follows:

    • Denautration 98˚C - 30 seconds
    • 25-30 cycles:
Step Temperature Time
Denature 98˚C 10 Seconds
Anneal 50-65 ˚C (depends on the Tm of primers) 30 Seconds
Extension 72˚C Depends on primer length 1 minute per Kb
Final Extension 72˚C 10 minutes
Hold 4˚C Hold

References
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