Reverse transcription polymerase chain reaction
Combined RT-PCR and qPCR are routinely used for analysis of gene expression and quantification of viral RNA in research and clinical settings.
[5] 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.
[19] On the other hand, the entire reaction from cDNA synthesis to PCR amplification occurs in a single tube in the one-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.
[23] The measurement approaches of end-point RT-PCR requires the detection of gene expression levels by the use of fluorescent dyes like ethidium bromide,[24][25] P32 labeling of PCR products using phosphorimager,[26] or by scintillation counting.
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.
[42] 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.
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.
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.
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.
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.
[47] 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.
[48] A simple method for elimination of false positive results is to include anchors, or tags, to the 5' region of a gene specific primer.
[49] 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.
One-step RT-PCR subjects mRNA targets (up to 6 kb) to reverse transcription followed by PCR amplification in a single test tube.
Next, place the PCR tube into a thermal cycler for one cycle wherein annealing, extending, and inactivating of reverse transcriptase occurs.
[53] 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 data, not only do reviewers have a difficult time evaluating these manuscripts, but the studies also become impossible to replicate.
[54] Recognizing the need for the standardization of the reporting of experimental conditions, the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE, pronounced mykee) guidelines have been published by an international consortium of academic scientists.
[53] The guideline consists of the following elements: 1) experimental design, 2) sample, 3) nucleic acid extraction, 4) reverse transcription, 5) qPCR target information, 6) oligonucleotides, 7) protocol, 8) validation, and 9) data analysis.
