Conclusion First:
All four techniques-PCR, qPCR, RT-PCR, and RT-qPCR-are based on the polymerase chain reaction (PCR), primarily differing in their application fields and detection targets.
- PCR is mainly used for the amplification and detection of DNA sequences.
- qPCR allows for real-time quantitative detection of DNA or cDNA content.
- RT-PCR is primarily used for the amplification and detection of RNA molecules.
- RT-qPCR combines RT-PCR and qPCR techniques to quantitatively analyze RNA molecules.
▆ I. Standard PCR
Standard PCR, also known as first-generation PCR, uses double-stranded DNA as a template and dNTP as a substrate to specifically amplify double-stranded DNA. Agarose gel electrophoresis is then used for qualitative analysis of the products.
The PCR process can be roughly divided into three basic steps: high-temperature denaturation (around 95℃), low-temperature annealing (between 55 and 60℃), and moderate-temperature extension (around 72℃, the optimal reaction temperature for DNA polymerase).
After several cycles of denaturation, annealing, and extension, the target gene can be amplified millions of times. Gel electrophoresis can be used to detect the amplified DNA (separating DNA based on its molecular weight for identification and purification).
▆ II. Quantitative Real-time PCR (qPCR)
Quantitative Real-time PCR, the second-generation PCR, collects real-time fluorescent signals from fluorescent probe-labeled target genes during the extension phase of the PCR amplification process. The copy number or expression level of the target gene is determined through the relationship between three parameters: fluorescent signal, Ct value, and initial concentration of the target gene. However, since its absolute quantitative analysis results depend on Ct values and standard curves, the so-called "quantification" is relative in a sense. Moreover, its detection sensitivity, accuracy, and resolution are limited under conditions of subtle differences in low-copy target gene template concentrations. The two commonly used fluorescence methods in qPCR are TaqMan probe and SYBR Green.
▆ III. Reverse Transcription PCR (RT-PCR)
Reverse Transcription PCR (RT-PCR) involves reverse transcribing an RNA strand into complementary DNA (using RNA-dependent DNA polymerase, or reverse transcriptase), which is then used as a template for DNA amplification through PCR (using deoxynucleotide primers and DNA-dependent DNA polymerase). Each cycle then doubles the amount, following standard PCR protocols. The template RNA can be total RNA, mRNA, or in vitro transcribed RNA products.
▆ IV. Real-time Reverse Transcription PCR (RT-qPCR)
Real-time Reverse Transcription PCR (RT-qPCR) combines RT-PCR and qPCR techniques to quantitatively analyze RNA molecules. RT-qPCR is widely used in RNA expression level studies, such as gene expression analysis and viral load detection.
