Reverse transcription is a technique used by researchers to generate a complementary strand of DNA (cDNA) from RNA. The technology is based on a retroviral mechanism whereby the enzyme reverse transcriptase can reverse transcribe RNA into DNA. This is especially helpful when scientists only have tissue and want to study gene sequence. In this situation, researchers can isolate mRNA from the tissue and then use reverse transcription to produce cDNA.
This article explores the foundation of reverse transcription technology, cDNA synthesis and downstream applications using reverse transcriptase.
Reverse Transcriptase Overview
Basic Science behind the Reverse Transcriptase Enzyme
In nature, the reverse transcriptase enzyme is what allows retroviruses (RNA type virus) to duplicate and integrate their RNA genomes into a host DNA genome. The enzyme has three biochemical activities enabling this process: RNA-dependent DNA polymerase activity, ribonuclease H activity and DNA-dependent DNA polymerase activity (Bhagavan & Ha, 2015).
Once in the host, viral RNA and its accompanying enzymes use host nucleotides to assemble a complementary single strand of DNA that is hybridized with the original RNA strand. RNase H cleaves the RNA-DNA hybrid, enabling formation of double-stranded DNA using DNA-dependent DNA polymerase. With the assistance of the enzyme integrase, the new strand of DNA is incorporated into the host genome (Bhagavan & Ha, 2015).
The Use of Reverse Transcriptase in Molecular Biology
The mechanism behind reverse transcription has expanded the world of molecular biology by helping scientists overcome earlier obstacles by allowing scientists to use RNA as starting material instead of DNA.
From the cDNA products, we are able to examine the genetic makeup of different
tumors, PCR traditionally and quantitatively,
express unique proteins,
generate libraries of DNA sequences that code important proteins, and more.
Viral Sources of Reverse Transcriptase
- Moloney murine leukemia virus (M-MLV): M-MLV demonstrates high efficiency when generating full-length cDNA using a long RNA sequence (>5 kb). This is because M-MLV reverse transcriptase has reduced RNase H activity. RNase H is an enzyme that is responsible for cleaving RNA from the RNA-DNA hybrid in cDNA synthesis, allowing the second strand of DNA to be produced by DNA polymerase.
Wildtype M-MLV has a lower reaction temperature that makes it challenging to perform reverse transcription on RNA with strong secondary structure. However, variants exist that further reduce RNase H activity. Compared to wildtype M-MLV, the H minus (H-) variant is more thermostable, allowing for a much higher reaction temperature (55°C).
M-MLV reverse transcriptase is ideal for cDNA and first strand cDNA synthesis, RT-PCR and gene expression validation using reverse transcription PCR (RT-PCR).
General Characteristics of M-MLV RT:
- Size: 70 kDa
- RNase H Activity: Reduced RNase H activity
- Reaction Temperature: up to 55°C
- Benefits: Greatly reduced RNase H activity, high temperature tolerance, ideal for full-length cDNA production
- Avian myeloblastosis virus (AMV): AMV, a commonly used reverse transcriptase in biotechnology, is suitable for cDNA and first strand cDNA synthesis, and RNA sequencing.
Because AMV reverse transcriptase can withstand higher temperatures, it is often used when RNA has stronger secondary structure. However, higher reaction temperatures can denature RNA. Some protocols combat the issue of overcoming strong secondary structure without jeopardizing RNA by incorporating a fast denaturing and cooling step.
AMV reverse transcriptase naturally has RNase H activity, which degrades RNA from the RNA/DNA hybrid.
General Characteristics of AMV RT:
- Size: 65 kDa α subunit, 95 kDa β subunit
- RNase H Activity: RNase H +
- Reaction Temperature: 25°C - 58°C. Optimal between 42°C - 48°C
- Benefits: Used with RNA that has strong secondary structure.
Choosing a Reverse Transcriptase for Your Experiment
Different reverse transcriptases are suited for different situations. This section highlights a few applications and the best choice transcriptase.
- Routine RT-PCR – for general RT-PCR, a standard enzyme should work just fine.
- Working with RNA with complex secondary structure – Consider using an AMV reverse transcriptase.
- Constructing cDNA libraries using long RNA – Use M-MLV reverse transcriptase with reduced RNase H activity.
- Performing one-step RT-PCR or RT-qPCR – Most kits available will use an M-MLV reverse transcriptase with reduced RNase H activity or a proprietary version of this RT. When performing one-step RT-PCR or RT-qPCR, M-MLV reverse transcriptase with the reduced RNase H activity will generally be a good choice.
Should I Do a One-Step or Two-Step RT-PCR/RT-qPCR?
There are some simple ways to help narrow down which method you should choose for either RT-PCR or RT-qPCR, and it all boils down to sensitivity requirements, experimental size and complexity, and how much available time you have. There are several kits on the market which include a reverse transcriptase suited for one-step and two-step processes.
- One-step PCR processes have many advantages. They give consistent results, the protocol is simple and fast, and there is less pipetting. This technique is, however, less sensitive and it’s not possible to optimize reactions individually. It also does not provide stock cDNA.
Choose one-step RT-PCR when:
- Running high-throughput experiments that require you to work fast
- Lower sensitivity is less concerning
- Working with many RNA samples
- You do not need stock cDNA
- Two-step PCR processes are extremely flexible and allow better control of your experiment. This technique allows individual optimization of cDNA synthesis and the PCR reaction. It’s more sensitive compared to the one-step method. Because random primers are used, the two-step method often runs more efficiently. And it provides stock cDNA that allows for aliquoting and use in multiple assays. The two step method does take much longer to perform and involves significantly more pipetting.
Choose two-step RT-PCR when:
- Running smaller experiments, and time is more available
- Your experiment requires higher sensitivity
- Working with fewer RNA samples
- You would like to generate stock cDNA for multiple analyses and assays
The Significance of cDNA
Use of cDNA in Biotechnology
The central dogma of biology states that genetic information is passed first from DNA, then to RNA and then used for protein production. Reverse transcription and cDNA synthesis enables scientists to work backward, decoding vital information about proteins and protein mutations. The value, however, of cDNA goes beyond that.
Researchers are able to use cDNA for RNA quantitation, to protect the genetic makeup of an endangered species, dive deeper into clinical research, understand the mRNA and protein involved during a given developmental stage, and so much more.
By being able to created cDNA libraries, scientists are able to study sequences specific to a given tissue and develop sharable databases.
cDNA libraries are based on mRNA complements, and represent the mRNA makeup within a given cell or tissue. Libraries provide a lot of information about the identity and functionality of specific genes. Libraries also provide proportional insights into the abundance of RNA produced in a given cell or tissue because the more an mRNA is expressed, the more cDNA will be produced and vice versa.
cDNA libraries are different from genomic libraries in the following ways:
- mRNA is the starting material for cDNA libraries. Therefore, cDNA libraries will only have sequences for the mRNA of a particular tissue.
- A genomic library might have a clone for all of an organism’s genes. A cDNA library will not.
- cDNA libraries provide information about the expression levels of mRNA. A genomic library will only offer us information on gene representation as they occur in the chromosome.
A benefit of cDNA and cDNA libraries, which is another point of separation from genomic libraries, is that cDNA does not have introns. This is extremely useful when using prokaryotic organisms for cloning since they do not have splicing capabilities.
cDNA Library Screening
Just as a traditional library might have a book of interest, a cDNA library will hold copies of a gene of interest, and researchers need a way of identifying that gene.
There are many colonies on a master plate of a cDNA library since the library holds the mRNA representation of a given tissue or cell. This is where library screening comes into play. Here are a few screening processes highlighted from the highly rated Shomu’s Biology on Youtube:
- Colony Hybridization: This technique identifies the DNA of interest using a radiolabeled probe that binds to the target sequence. Hybridization will be useful when you know the sequence of the gene you’re interested in.
In this screening method, a nylon filter paper is used to replicate a master plate containing colonies (each colony contains a homogenous population of identical closed plasmid) by pressing it onto the master plate thereby transferring cells from the colonies from the master plate onto the nylon transfer paper. The filter paper is treated with an alkaline solution to lyse the cells and denature the DNA. Then, radio labeled probes comprising complementary oligos of the target sequence are added. The probes hybridize with DNA from the lysed cells. Then, the filter paper is exposed to X-ray which will once developed, will allow visualization of the target, and enable us to make a comparison between the labeled nylon paper and the master plate to find the colonies containing our DNA of interest. The selected colonies are then picked and grown on nutrient medium.
- Screening with Labeled Oligonucleotide Probes: This method is going to be useful when you do not know the DNA sequence. It is similar in many ways to colony hybridization; however, this technique relies on the peptide sequence rather than the DNA sequence. From the peptide sequence, a researcher can find the possible DNA sequence, produce a complementary sequence and use that as a probe/primer. From there, the rest of the technique follows colony hybridization.
- Immunological Screening: This process uses primary and secondary antibodies to identify colonies of interest. Here, nitrocellulose paper is laid on the petri dish where proteins will bind. The nitrocellulose is first incubated with the primary antibody, then with a radiolabeled secondary antibody. After development, the relative location of the colony of interest can be determined using the exposed X-ray film as a guide.
- Subtractive Screening: The previous methods allow researchers to start with some kind of known in order to screen their library. Subtractive screening, however, is capable of identifying novel gene expression from mRNA. For example, exposure to a new drug treatment or environmental contaminant is believed to induce unique gene expression from a control. Subtractive screening (also called differential screening) helps examine this unique expression and ultimately allows researchers to compare the DNA in question against a control to find a difference in expression.
Applications Involving cDNA and Reverse Transcription
Reverse transcription is the key to obtaining the initial
DNA (cDNA) which can then be used in a number of applications to further study
- RT-PCR (reverse transcription polymerase chain reaction) – Simply put, this is PCR where RNA is the starting material. Using reverse transcriptase, the mRNA is reverse transcribed into cDNA which can then be used as a template for PCR amplification.
There are options for traditional RT-PCR kits and for One Step kits. One Step RT-PCR kits are extremely easy to use, involving a reaction setup with an RNA template, reverse transcriptase and PCR mix, so that cDNA is both synthesized and amplified.
- RT-qPCR (Reverse transcription quantitative PCR) – Just like RT-PCR, the definition of RT-qPCR is simply quantitative PCR involving RNA as the initial starting material. This method can be done either in one step or two.
One Step RT-qPCR features more consistent results, fewer steps, and ideal for high-throughput amplifications. One of the major downsides is the inability to individualize and optimize each process (cDNA synthesis and PCR). The one step process can also have reduced sensitivity.
Performing RT-qPCR in two steps (cDNA synthesis followed by qPCR) allows better experimental optimization.
Synthesized cDNA also allows researchers to perform protein
purification and expression, and gene expression profiling.
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