Tanuka Biswas is a science communication professional, with over a decade of experience in molecular biology, cancer biology, and content strategy. She earned her PhD in biomedical sciences at the University of Texas Health Science Center, with a focus on cancer biology. She is passionate about making complex scientific concepts accessible to readers.
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Quantitative polymerase chain reaction (qPCR) is a molecular technique that has reshaped the landscape of biological research and medicine due to its high sensitivity and specificity.
qPCR, also known as real-time PCR, is an extension of traditional PCR, where DNA amplification over time is quantitated by measuring fluorescence from dyes intercalating into nascent DNA strands or by cleavage of target-specific probes. Therefore, the method combines detection with amplification, providing researchers with crucial data in real time.1
Like PCR, qPCR amplifies DNA using a DNA polymerase enzyme. This adds new nucleotides to the 3’-OH end of a short, single-stranded DNA segment, called a primer, which complements the template strand.2 The reaction occurs in specialized thermal cyclers equipped with fluorometers that measure fluorescent signals, quantifying both relative and absolute DNA amounts when combined with reference values.
Reverse transcription PCR (RT-PCR) is often confused with real-time PCR. RT-PCR utilizes a reverse transcriptase enzyme to convert ribonucleic acid (RNA) into complementary DNA (cDNA).1 When combined with qPCR, the method is called reverse transcription real-time quantitative PCR (RT-qPCR).3 RT-qPCR is a powerful tool because it enables researchers to measure gene expression of specific genes using RNA as the template.
Real-time monitoring, high sensitivity, and specificity enable the precise measurement of genetic material and render qPCR invaluable for various applications. Some key examples are listed below.
A typical qPCR reaction starts with a master mix containing DNA polymerase, deoxynucleotide triphosphates (dNTPs), primers, a dye or reporter, and template DNA.4 The reaction runs in a thermal cycler, programmed for approximately 35-40 amplification cycles, preceded by an initial denaturation phase.
Scientists can employ several detection methods in qPCR experiments, depending on the specific application and the nature of the target molecule. Some common qPCR detection methods are described in the table below.
Table 1: Common qPCR detection methods with their pros and cons
Dyes are versatile and cost-effective but have low specificity.
The probes are highly specific but require precise and custom design.
While highly versatile and specific, these can be complex to design.
Locked nucleic acid (LNA) probes
While their specificity and thermal stability ensure precision in measurement, designing these probes often requires extensive optimization.
Appropriate controls in a qPCR assay ensure reliability and accuracy and help track any potential issues while providing a baseline for relative quantification.
Some important controls include the following.
Successful qPCR primer design is pivotal for specific and efficient amplification. Detailed below are some important considerations to bear in mind.6
Analyzing results from qPCR centers on an amplification plot, which charts the cycle number against the fluorescent signal. Initial qPCR cycles with minimal amplification and relatively low fluorescent signals form a baseline for analysis. At a specific cycle number, known as the threshold cycle (Ct), the fluorescent signal exceeds the baseline.
Scientists can quantify qPCR results using either absolute or relative quantification. Absolute quantification determines the total target DNA or RNA in experimental samples based on a standard plot. Relative quantification determines changes in the sample relative to a reference, typically an untreated control, utilizes endogenous genes with consistent expression, and can be measured using a standard curve7 or a comparative Δ Δ Ct method.8
Δ Δ Ct = Δ Ct (treated sample) − Δ Ct (untreated control)
Δ Ct (treated sample) = Ct (target gene in treated) − Ct (reference gene in treated)
Δ Ct (untreated control) = Ct (target gene in untreated) − Ct (reference gene in untreated)
In conclusion, qPCR is a pivotal technique in molecular biology, enabling precision nucleic acid quantification and supporting various applications such as genetics, diagnostics, and beyond.
Tanuka Biswas is a science communication professional, with over a decade of experience in molecular biology, cancer biology, and content strategy. She earned her PhD in biomedical sciences at the University of Texas Health Science Center, with a focus on cancer biology. She is passionate about making complex scientific concepts accessible to readers.
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