RT-qPCR: Application & Overview

RT-qPCR: Overview

The development of polymerase chain reaction (PCR) brought Kary Mullis a Nobel prize in chemistry in 1992. The high-throughput technique has revolutionized research in molecular biology by allowing researchers to create many copies of the target sequence or gene of interest through PCR assays

Based on the application or RNA samples that need to work on, PCR has many types:

  • Multiplex PCR
  • Long Range PCR
  • Hot-Start PCR
  • High-Fidelity PCR
  • Real-Time RT-PCR
  • Nested PCR

In quantitative RT-PCR (also known as RT-qPCR or quantitative reverse transcription PCR), the target RNA sequence is detected and amplified by the combined power of reverse transcription and quantitative PCR or real-time PCR. It allows the real-time analysis of amplified DNA using fluorescent dyes during the usual 40 PCR cycles of the procedure. 

One common approach to quantifying DNA in RT-qPCR is by using either a hybridization probe, double-stranded DNA binding dye such as SYBR® Green dye, or hydrolysis probes such as TaqMan® probes. More substrates and ligands can be used to amplify the fluorescent signal for easy detection and quantification.

It’s essential to choose a reverse transcriptase enzyme with high thermal stability for the RT-qPCR process. This is because the enzyme allows cDNA synthesis at a higher temperature, maintaining high levels of the secondary structure during RNA transcription.

RT-qPCR acts as a primary tool in a spectrum of lab applications, including RNA interference (RNAi) validation, gene expression level quantification, and pathogen detection. 

In this article, we will learn more about RT-qPCR, its procedure, its roles in different lab assays, and the industries involved in RT-qPCR applications.

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How Does RT-qPCR Work?

In PCR reaction, the DNA polymerase enzyme uses sequence-specific Primers, dNTP, and the DNA polymerase enzyme for the synthesis of a new DNA strand on the single-stranded DNA template (or DNA amplicon). The same reaction occurs in RT-qPCR, however, with an additional step involving reverse transcriptase.

cDNA synthesis in a thermal cycler through RT-qPCR consists of four steps:

  • Pre-denaturation: An optional step and only performed when the RNA template has too many secondary structures. It’s carried out after RNA extraction and sample preparation steps.
  • Primer Extension: Annealing of primer and extending using polymerase enzyme and dNTPs.
  • cDNA synthesis: Formation of a complementary DNA strand (PCR product) by reverse transcriptase enzyme using an RNA template.
  • Reaction Termination: At this step, qPCR inhibition by active reverse transcriptase is prevented.
Figure: RT-qPCR steps from sample isolation to Data analysis.

Some essential components of RT-qPCR are PCR Primers, reverse transcriptase enzymes, PCR master mix, Taq DNA polymerase enzyme, RNA samples (such as total RNA or viral genomic RNA), dNTPs, genomic RNA, and DNase and RNase inhibitors.

Conditions for PCR reaction at each step include:

  • Enzyme activation step: At 95°C for 15 minutes
  • Denaturation: Followed by 40 cycles consisting of 15 seconds at 95°C
  • Annealing: At 58–64°C (Based on primer) for 30 seconds 
  • Elongation: At 72°C for 30 seconds 

Melting curve analysis and agarose gel electrophoresis can be used to verify the specificity of amplified PCR products. A standard curve for each qPCR reaction can be generated after serial dilution of a standard cDNA.

Types of RT-qPCR Assay

During RT-qPCR, RNA templates (such as total RNA or mRNA) are used as a starting material, which is converted to a cDNA by the enzyme reverse transcriptase. For the quantification of cDNA or gene expression, two methods of RT-qPCR are available:

  • One-Step RT-qPCR: Here, cDNA synthesis or reverse transcription and qPCR reactions are carried out in a single tube using sequence-specific primers for amplification of the target sequence. It’s mainly used for high-throughput and diagnostic applications.
  • Two-Step RT-qPCR: Here, reverse transcriptase is first used to convert RNA into cDNA. Then, the resulting cDNA is used as a template for multiple qPCR reactions. It allows more flexibility in choosing PCR reagents or master mix and reverse transcriptase enzymes for improved amplification efficiency. However, it requires more optimization than a one-step approach.
Figure: Two methods of RT-qPCR.

What is RT-qPCR Used For?

RT-qPCR is one of the most commonly used tools in molecular biology labs to analyze and quantify nucleic acids in diverse biological samples quickly and accurately. Its range of applications includes the detection of genetically modified organisms in food (through cDNA cloning), gene expression analysis, and cancer phenotyping. 

Gene Expression Quantification

qPCR assays are widely used in labs to quantitatively detect mutant genes or copy numbers of the gene in transformed cell lines. When used in combination with reverse transcriptase, the technique can analyze changes in gene expression. For example, it can measure changes in cellular mRNA levels, which indicate an increase or decrease in gene expression level during drug treatment under different environmental conditions. 

Pathogen Detection

RT-qPCR has a massive application in detecting pathogens, such as viruses, and diagnosis of infectious diseases. The technique was commonly used during the coronavirus pandemic to detect the SARS-CoV-2 virus in the samples of the patients. It’s still the gold standard for COVID-19 tests.

Further, the technique is also widely used for the detection, quantification, and typing of different microbial agents in clinical diagnostics, veterinary assessments, and food safety.

Figure: Steps to detect Coronavirus in any given samples using RT-qPCR.

RNAi Validation

RT-qPCR is used to verify the loss of function of selective genes due to RNAi. It’s exploited as a primary analytical tool to quantify the difference in gene expression level caused by RNA interference.

GMO Detection

Due to its robustness and sensitivity, RT-qPCR is considered the gold standard for the detection and quantification of genetically modified organisms (GMOs). Further, it can be used to introduce specific characteristics into the modified organisms.

What Industries Use RT-qPCR? 

PCR experiments are performed in a range of healthcare, medical, biotechnology, and pharma industries. They help in the detection and isolation of pathogenic organisms, study viruses, and diagnose several microbial or viral diseases.

Healthcare

In the healthcare sector, RT-qPCR experiments are used to identify and quantify microbial agents in different edible samples with food safety concerns. Further, it has been widely used to diagnose viral diseases, such as COVID-19. 

Pharmaceutical

With its capability to detect and quantify, qPCR is a versatile and widely applied laboratory technique for both diagnostic and basic research applications. It’s also one of the frequently used techniques in drug discovery and cancer phenotyping.

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RT-qPCR is one of the most commonly used molecular biology lab assays to amplify RNA. Here, the RNA samples are collected and converted to cDNA, which is amplified by PCR for further applications. Based on the number of tubes used in the experiment, RT-qPCR is classified into one-step and two-step approaches.

The high-throughput RT-qPCR workflow is mainly used for the detection and isolation of pathogens, disease diagnosis, cancer phenotyping, and drug discovery. To perform such advanced studies, you need high-quality lab-grade reagents paired with high-tech equipment.

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