Laboratory technique to multiply an RNA sample for study
Reverse transcription polymerase chain reaction (RT-PCR) is a laboratory technique combining reverse transcription of RNA into DNA (in this context called complementary DNA or cDNA) and amplification of specific DNA targets using polymerase chain reaction (PCR). It is primarily used to measure the amount of a specific RNA. This is achieved by monitoring the amplification reaction using fluorescence, a technique called real-time PCR or quantitative PCR (qPCR). Confusion can arise because some authors use the acronym RT-PCR to denote real-time PCR. In this article, RT-PCR will denote Reverse Transcription PCR. Combined RT-PCR and qPCR are routinely used for analysis of gene expression and quantification of viral RNA in research and clinical settings.
The close association between RT-PCR and qPCR has led to metonymic use of the term qPCR to mean RT-PCR. Such use may be confusing, as RT-PCR can be used without qPCR, for example to enable molecular cloning, sequencing or simple detection of RNA. Conversely, qPCR may be used without RT-PCR, for example, to quantify the copy number of a specific piece of DNA.
Nomenclature.
The combined RT-PCR and qPCR technique has been described as quantitative RT-PCR or real-time RT-PCR (sometimes even called quantitative real-time RT-PCR), has been variously abbreviated as qRT-PCR, RT-qPCR, RRT-PCR, and rRT-PCR. In order to avoid confusion, the following abbreviations will be used consistently throughout this article:
Not all authors, especially earlier ones, use this convention and the reader should be cautious when following links. RT-PCR has been used to indicate both real-time PCR (qPCR) and reverse transcription PCR (RT-PCR).
History.
Since its introduction in 1977, Northern blot has been used extensively for RNA quantification despite its shortcomings: (a) time-consuming technique, (b) requires a large quantity of RNA for detection, and (c) quantitatively inaccurate in the low abundance of RNA content. However, since PCR was invented by Kary Mullis in 1983, RT PCR has since displaced Northern blot as the method of choice for RNA detection and quantification.
RT-PCR has risen to become the benchmark technology for the detection and/or comparison of RNA levels for several reasons: (a) it does not require post PCR processing, (b) a wide range ( ;107-fold) of RNA abundance can be measured, and (c) it provides insight into both qualitative and quantitative data. Due to its simplicity, specificity and sensitivity, RT-PCR is used in a wide range of applications from experiments as simple as quantification of yeast cells in wine to more complex uses as diagnostic tools for detecting infectious agents such as the avian flu virus and SARS-CoV-2.
Principles.
In RT-PCR, the RNA template is first converted into a complementary DNA (cDNA) using a reverse transcriptase (RT). The cDNA is then used as a template for exponential amplification using PCR. The use of RT-PCR for the detection of RNA transcript has revolutionized the study of gene expression in the following important ways:
One-step RT-PCR vs two-step RT-PCR.
The quantification of mRNA using RT-PCR can be achieved as either a one-step or a two-step reaction. The difference between the two approaches lies in the number of tubes used when performing the procedure. The two-step reaction requires that the reverse transcriptase reaction and PCR amplification be performed in separate tubes. The disadvantage of the two-step approach is susceptibility to contamination due to more frequent sample handling. On the other hand, the entire reaction from cDNA synthesis to PCR amplification occurs in a single tube in the one-step approach. The one-step approach is thought to minimize experimental variation by containing all of the enzymatic reactions in a single environment. It eliminates the steps of pipetting cDNA product, which is labor-intensive and prone to contamination, to PCR reaction. The further use of inhibitor-tolerant thermostable DNA polymerases, polymerase enhancers with an optimized one-step RT-PCR condition, supports the reverse transcription of the RNA from unpurified or crude samples, such as whole blood and serum. However, the starting RNA templates are prone to degradation in the one-step approach, and the use of this approach is not recommended when repeated assays from the same sample is required. Additionally, the one-step approach is reported to be less accurate compared to the two-step approach. It is also the preferred method of analysis when using DNA binding dyes such as SYBR Green since the elimination of primer-dimers can be achieved through a simple change in the melting temperature. Nevertheless, the one-step approach is a relatively convenient solution for the rapid detection of target RNA directly in biosensing.
End-point RT-PCR vs real-time RT-PCR.
Quantification of RT-PCR products can largely be divided into two categories: end-point and real-time. The use of end-point RT-PCR is preferred for measuring gene expression changes in small number of samples, but the real-time RT-PCR has become the gold standard method for validating quantitative results obtained from array analyses or gene expression changes on a global scale.
End-point RT-PCR.
The measurement approaches of end-point RT-PCR requires the detection of gene expression levels by the use of fluorescent dyes like ethidium bromide, P32 labeling of PCR products using phosphorimager, or by scintillation counting. End-point RT-PCR is commonly achieved using three different methods: relative, competitive and comparative.
Real-time RT-PCR.
The emergence of novel fluorescent DNA labeling techniques in the past few years has enabled the analysis and detection of PCR products in real-time and has consequently led to the widespread adoption of real-time RT-PCR for the analysis of gene expression. ;ref name="lww/cmj/2021/09050/rt-rt-pc.5" ; ;/ref ; Not only is real-time RT-PCR now the method of choice for quantification of gene expression, it is also the preferred method of obtaining results from array analyses and gene expressions on a global scale. ;ref name="iaea/covid_rt-rt-pcr" ; ;/ref ; Currently, there are four different fluorescent DNA probes available for the real-time RT-PCR detection of PCR products: SYBR Green, TaqMan, molecular beacons, and scorpion probes. All of these probes allow the detection of PCR products by generating a fluorescent signal. While the SYBR Green dye emits its fluorescent signal simply by binding to the double-stranded DNA in solution, the TaqMan probes', molecular beacons' and scorpions' generation of fluorescence depend on Förster Resonance Energy Transfer (FRET) coupling of the dye molecule and a quencher moiety to the oligonucleotide substrates.
Two strategies are commonly employed to quantify the results obtained by real-time RT-PCR; the standard curve method and the comparative threshold method.
Application.
The exponential amplification via reverse transcription polymerase chain reaction provides for a highly sensitive technique in which a very low copy number of RNA molecules can be detected. RT-PCR is widely used in the diagnosis of genetic diseases and, semiquantitatively, in the determination of the abundance of specific different RNA molecules within a cell or tissue as a measure of gene expression.
Research methods.
RT-PCR is commonly used in research methods to measure gene expression. For example, Lin et al. used qRT-PCR to measure expression of Gal genes in yeast cells. First, Lin et al. engineered a mutation of a protein suspected to participate in the regulation of Gal genes. This mutation was hypothesized to selectively abolish Gal expression. To confirm this, gene expression levels of yeast cells containing this mutation were analyzed using qRT-PCR. The researchers were able to conclusively determine that the mutation of this regulatory protein reduced Gal expression. Northern blot analysis is used to study the RNA's gene expression further.
Gene insertion.
RT-PCR can also be very useful in the insertion of eukaryotic genes into prokaryotes. Because most eukaryotic genes contain introns, which are present in the genome but not in the mature mRNA, the cDNA generated from a RT-PCR reaction is the exact (without regard to the error-prone nature of reverse transcriptases) DNA sequence that would be directly translated into protein after transcription. When these genes are expressed in prokaryotic cells for the sake of protein production or purification, the RNA produced directly from transcription need not undergo splicing as the transcript contains only exons. (Prokaryotes, such as E. coli, lack the mRNA splicing mechanism of eukaryotes).
Genetic disease diagnosis.
RT-PCR can be used to diagnose genetic disease such as Lesch–Nyhan syndrome. This genetic disease is caused by a malfunction in the HPRT1 gene, which clinically leads to the fatal uric acid urinary stone and symptoms similar to gout. Analyzing a pregnant mother and a fetus for mRNA expression levels of HPRT1 will reveal if the mother is a carrier and if the fetus will likely to develop Lesch–Nyhan syndrome.
Cancer detection.
Scientists are working on ways to use RT-PCR in cancer detection to help improve prognosis, and monitor response to therapy. Circulating tumor cells produce unique mRNA transcripts depending on the type of cancer. The goal is to determine which mRNA transcripts serve as the best biomarkers for a particular cancer cell type and then analyze its expression levels with RT-PCR.
RT-PCR is commonly used in studying the genomes of viruses whose genomes are composed of RNA, such as Influenzavirus A, retroviruses like HIV and SARS-CoV-2.
Challenges.
Despite its major advantages, RT-PCR is not without drawbacks. The exponential growth of the reverse transcribed complementary DNA (cDNA) during the multiple cycles of PCR produces inaccurate end point quantification due to the difficulty in maintaining linearity. In order to provide accurate detection and quantification of RNA content in a sample, qRT-PCR was developed using fluorescence-based modification to monitor the amplification products during each cycle of PCR. The extreme sensitivity of the technique can be a double-edged sword since even the slightest DNA contamination can lead to undesirable results. A simple method for elimination of false positive results is to include anchors, or tags, to the 5' region of a gene specific primer. Additionally, planning and design of quantification studies can be technically challenging due to the existence of numerous sources of variation including template concentration and amplification efficiency. Spiking in a known quantity of RNA into a sample, adding a series of RNA dilutions generating a standard curve, and adding in a no template copy sample (no cDNA) may used as controls.
Protocol.
RT-PCR can be carried out by the one-step RT-PCR protocol or the two-step RT-PCR protocol.
One-step RT-PCR.
One-step RT-PCR subjects mRNA targets (up to 6 kb) to reverse transcription followed by PCR amplification in a single test tube. Using intact, high-quality RNA and a sequence-specific primer will produce the best results.
Once a one-step RT-PCR kit with a mix of reverse transcriptase, Taq DNA polymerase, and a proofreading polymerase is selected and all necessary materials and equipment are obtained a reaction mix is to be prepared. The reaction mix includes dNTPs, primers, template RNA, necessary enzymes, and a buffer solution. The reaction mix is added to a PCR tube for each reaction, followed by template RNA. The PCR tubes are then placed in a thermal cycler to begin cycling. In the first cycle, the synthesis of cDNA occurs. The second cycle is the initial denaturation wherein reverse transcriptase is inactivated. The remaining 40-50 cycles are the amplification, which includes denaturation, annealing, and elongation. When amplification is complete, the RT-PCR products can be analyzed with gel electrophoresis.
Two-step RT-PCR.
Two-step RT-PCR, as the name implies, occurs in two steps. First the reverse transcription and then the PCR. This method is more sensitive than the one-step method. Kits are also useful for two-step RT-PCR. Just as for one-step PCR, use only intact, high-quality RNA for the best results. The primer for two-step PCR does not have to be sequence-specific.
Step one.
First combine template RNA, primer, dNTP mix, and nuclease-free water in a PCR tube. Then, add an RNase inhibitor and reverse transcriptase to the PCR tube. Next, place the PCR tube into a thermal cycler for one cycle wherein annealing, extending, and inactivating of reverse transcriptase occurs. Finally, proceed directly to step two which is PCR, or store product on ice until PCR can be performed.
Step two.
Add master mix which contains buffer, dNTP mix, MgCl2, Taq polymerase, and nuclease-free water to each PCR tube. Then add the necessary primer to the tubes. Next, place the PCR tubes in a thermal cycler for 30 cycles of the amplification program. This includes denaturation, annealing, and elongation. The products of RT-PCR can be analyzed with gel electrophoresis.
Publication guidelines.
Quantitative RT-PCR assay is considered to be the gold standard for measuring the number of copies of specific cDNA targets in a sample but it is poorly standardized. As a result, while there are numerous publications utilizing the technique, many provide inadequate experimental detail and use unsuitable data analysis to draw inappropriate conclusions. Due to the inherent variability in the quality of any quantitative PCR