Blog:

The Basics of Reverse Transcription PCR (RT-PCR)

What is Reverse Transcription Polymerase Chain Reaction (RT-PCR)?

Reverse transcription-polymerase chain reaction is a molecular technique commonly called RT-PCR. PCR stands for polymerase chain reaction, a laboratory method used to amplify specific regions of DNA for diagnosis and analysis in medical research. Reverse transcriptase PCR, instead of DNA, uses mRNA as the starting template.

Real-time RT-PCR is a modified version of PCR in which the enzyme called reverse transcriptase is used. This enzyme facilitates the conversion of an RNA sequence into its complementary DNA sequence. RT-PCR works on this principle of reverse transcription. It is a real-time technique that combines reverse transcription of RNA into DNA and the amplification of target DNA. It’s a qualitative means of detecting a particular RNA sequence with a high degree of accuracy and sensitivity. 

Karry Mullis discovered RT-PCR to facilitate RNA detection and quantification. He won the Nobel Prize in Chemistry 1993 for introducing this technique and revolutionizing the field of molecular biology.

RT-PCR Applications

RT-PCR holds applications in: 

  • Genome sequencing
  • Detecting changes in gene or chromosomes
  • Characterization of genes
  • The study of gene expressions and mutations
  • RNA virus research
  • Diagnoses of diseases such as leukemia
  • The monitoring of infections.

A tremendous contribution of RT-PCR is seen in the diagnosis of the RNA-virus SARS-CoV-2. It became the benchmark technology for detection of specific RNA sequences and it was because of this sensitive in vitro method that mass diagnosis of coronavirus became a possibility.

In this article, we’ll review the real-time reverse transcription polymerase chain reaction (RT-PCR) process and how it varies from other types of PCR techniques.

Excedr’s lease program supports laboratories on a budget. Lease the cutting-edge lab equipment you need, accelerate your R&D, and achieve milestones quicker when working with Excedr.

RT-PCR Process

RT-PCR is a molecular diagnostic tool that works on the principle of converting the RNA template to a complementary DNA (cDNA) using the reverse transcriptase enzyme. This cDNA then undergoes exponential amplification using PCR to form multiple copies, which are then used for downstream analysis.

In a typical PCR, DNA is the template, and the enzyme used is Taq polymerase. However, in order to amplify RNA, reverse transcription needs to be carried out since RNA is not an efficient template for Taq polymerase. Hence, the modified version of PCR has an extra step of RNA being converted to DNA, then PCR is carried out.

To begin, a typical RT-PCR assay requires nucleic acids, primers, a reverse transcriptase enzyme, RNA sample, DNA polymerase, and an RT-PCR buffer.

Let’s have a detailed look at the RT-PCR process:

  • After RNA isolation is performed, the RNA is extracted.
  • An aliquot of extracted RNA sample is added to a reaction mixture. The reaction mixture consists of nucleotides, reverse transcriptase, and primers specific for the gene of interest.
  • Primers will anneal to the extracted RNA if the target is present.
  • Reverse transcriptase performs its function of synthesizing the complementary DNA (cDNA) strand.
  • The RNA/DNA hybrid now undergoes the steps of PCR:
    • Denaturation: At 95o C the RNA/DNA strands denature.
    • Annealing: Primers anneal to the newly formed cDNA
    • Extension: Extending from the primer, the polymerase synthesizes a new DNA strand by replication.
  • Multiple cycles in the thermal cycler increase the number of copies of DNA as PCR products.

Furthermore, RT-PCR can be carried out in two different ways:

1-Step RT-PCR

In this method, the reaction tube of reverse transcription and polymerase chain reaction is the same, and both processes are performed simultaneously. This simple and convenient one-step approach is used for performing a small number of assays. Since both reactions are conducted in a single reaction tube, the primers used are sequence-specific.

Advantages:

  • Quick setup
  • Shorter handling time
  • Minimal chances of contamination
  • Easy processing

Disadvantages:

  • There is a limit to optimizing both the reactions individually
  • Less sensitive 
  • To analyze new targets or repeat amplification, RNA aliquot needs to be saved

Source

2-Step RT-PCR

In this PCR reaction, the reverse transcription of RNA to cDNA is initially carried out between 40oC and 50oC. The resulting cDNA is then amplified in a separated reaction—cDNA synthesis and PCR amplification are uncoupled.

The reaction vessel, buffer, reagents, conditions, and priming strategies used in each step are different. Unlike the one-step RT-PCR, a mixture of random hexamers, gene-specific primers, and oligo-dT primers are used in two-step RT-PCR. This allows a higher yield of cDNA to be obtained which can either be stored or further amplified.

This is a highly sensitive but time-consuming approach.

Advantages:

  • RT primers can be of choice
  • From a single RNA source, multiple targets can be amplified
  • Can be performed even with limited starting material
  • Reaction optimization is flexible
  • Reverse transcriptases and DNA polymerase can be separately selected with greater flexibility

Disadvantages:

  • More machine time and setup
  • More optimization needed
  • Increased chances of contamination

Source

What is the Difference Between RT-PCR, qPCR, RT-qPCR?

RT-PCR

RT-PCR stands for reverse transcription PCR, not real-time PCR. It is a method that allows the generation of cDNA from RNA templates. The single-stranded cDNA is then converted to double-stranded cDNA and amplified.

RT-PCR is mainly used for molecular cloning of genes of interest (GOI).

qPCR

qPCR stands for quantitative real-time PCR. It is a quantitative method of analysis using the principle of a typical PCR. In this process, however, the measurement of DNA amplification is carried out in real-time instead of at the end of the process with an agarose gel.

Quantitation of PCR products is performed using fluorescent probes like intercalating dye or hydrolysis-based fluorescent probes. Quantitative PCR helps in the detection of pathogens and the determination of the copy number of the DNA sequence of interest.

Sample preparation: DNA free from any contamination is isolated.

RT-qPCR

RT-PCR coupled with qPCR gave rise to the method of RT-qPCR. Here, RT-PCR is the first step, followed by qPCR. Measurement of RNA levels can be accomplished using cDNA in a qPCR reaction. Moreover, model systems with inhibitors, stimulants, siRNA, or knock models are used to investigate gene expression changes. 

Sample preparation: The starting template is RNA, enabling RNA extraction to be carried out. The type of RNA extraction depends on the type of RNA required. The total RNA extraction kits isolate mRNA, tRNA, rRNA, ncRNA, and miRNA. It is important to note that the RNA should be free from any DNA contamination.

Using a virtual gel electrophoresis system for sample degradation is a preferred approach.

Mainly, two types of detection methods are used in qPCR and RT-PCR:

  1. Fluorescent dyes: These bind non-specifically to double-stranded DNA. A popular fluorescent dye is SYBR ® Green that intercalates with newly synthesized DNA. More fluorescence means more DNA produced.
  2. Hydrolysis probes: Probes like TaqMan depend on Förster Resonance Energy Transfer (FRET). These are specific sequences that bind downstream to qPCR primers, making them target-specific.

Quantitation and data analysis of the results from cycles of qPCR and RT-qPCR are performed with the help of an amplification curve with initiation, exponential, and plateau phases. 

This amplification curve is generated using a serial dilution of standards of known concentration. The analysis of the data can be based on absolute quantitation or relative quantitation.

Source

High-Quality Lab Equipment on Lease with Excedr

RT-PCR has become an increasingly important molecular biology technique widely used across the globe, especially after the outbreak of the COVID-19 virus. This method has given new hope to medical research for conducting disease diagnostics, molecular cloning, and recombinant DNA technology.

RT-PCR requires a proper setup and contamination-free environment for its conduction. Though the materials used might be similar, RT-PCR, qPCR, and RT-qPCR are different PCR methods altogether. 

A life science or medical laboratory is incomplete without a high-quality PCR setup. Excedr provides custom leasing solutions for all your lab needs. Whether it is PCR systems or any other clinical, biotech, or general lab equipment, Excedr has you covered. 

Contact us to learn more today.

Traditional equipment procurement methods don’t always make sense. If you’re struggling to fund an equipment acquisition, we can help. Lease the lab equipment you need without breaking the bank.