What is real-time quantitative PCR (qPCR)?
Nucleic acid amplification and detection technology is one of the most valuable tools in biological research today. Scientists in all fields of the life sciences—basic research, biotechnology, medicine, forensics, diagnostics, and more—use these methods in a wide range of applications. For some applications, qualitative nucleic acid detection is sufficient. However, other applications require quantitative analysis. Real-time PCR can be used for qualitative and quantitative analysis; selecting the best method for your application requires extensive technical knowledge. This section provides an overview of real-time PCR, reverse transcription quantitative PCR techniques, and the instrumentation options Bio-Rad offers for these techniques. Steps for RNA isolation such as sample collection, RNA extraction, and analysis of RNA quality and quantity are also provided.
What is real-time PCR?
In traditional PCR, amplified DNA products or amplicons are detected in an end-point assay. In real-time PCR, the accumulation of amplified product is measured in real time as the reaction progresses, and product quantification is performed after each cycle.
The following qPCR workflow describes the steps for real-time PCR. First, an amplification reaction is set up with PCR reagents and unique or custom primers. Reactions were then performed on a real-time PCR machine, and the collected data were analyzed with dedicated instrument software.
Real-time detection of PCR products can be achieved by adding fluorescent reporter molecules to each reaction well to generate more fluorescence as the amount of product DNA increases. Fluorescent chemistries used for this purpose include DNA-binding dyes and fluorescently labeled sequence-specific primers or probes. A dedicated thermal cycler equipped with a fluorescence detection module was used to monitor the amplification of the fluorescence signal. The measured fluorescence is proportional to the total amount of amplicons; the change in fluorescence over time is used to calculate the number of amplicons produced per cycle.
The main advantage of real-time quantitative PCR over PCR is that real-time quantitative PCR can accurately and highly sensitively determine the initial copy number of template DNA (the amplified target sequence) over a large dynamic range. Real-time PCR results can be qualitative (presence or absence of sequence) or quantitative (copy number). Real-time fluorescent quantitative PCR (Quantitative real-time PCR) is also called qPCR analysis. In contrast, PCR is at best semi-quantitative. In addition, real-time qPCR data can be evaluated without gel electrophoresis, reducing experimental time and increasing throughput. Finally, since real-time qPCR reactions are performed in a unified closed-tube qPCR system, data evaluation is performed in a unified closed-tube qPCR system, thus reducing the chance of contamination and eliminating post-amplification in qPCR analysis operational needs.
Professional Portable qPCR System Manufacturer
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