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The secrets of reverse transcription polymerase chain reaction: How to reveal gene expression through RNA?
Reverse transcription polymerase chain reaction (RT-PCR) is a laboratory technique that combines the transcription of RNA into DNA (in this context called complementary DNA or cDNA) and the amplification of a specific DNA target. This process uses the polymerase chain reaction (PCR). This technique is mainly used to measure the amount of specific RNA by monitoring the fluorescence of the amplification reaction, which is called real-time PCR or quantitative PCR (qPCR). In the scientific literature, RT-PCR is sometimes confusingly used by some authors to refer to real-time PCR. In this article, we refer to RT-PCR specifically as reverse transcription PCR.
Due to its simplicity, high specificity, and sensitivity, RT-PCR has been used in a wide variety of experiments, from quantifying yeast cells in wine to detecting infectious pathogens.
Principles and Applications of RT-PCR
The principle of RT-PCR is to first convert the RNA template into cDNA, which is then exponentially amplified using PCR. This revolutionary change in the process allows us to detect transcripts of nearly all genes and to amplify samples, eliminating the large amounts of starting material required when using Northern blotting.
RT-PCR has become one of the most important techniques from gene expression analysis to diagnosis of infectious pathogens. For example, scientists are studying how to use RT-PCR for cancer testing to improve prognosis and monitor treatment response.
For example, circulating tumor cells produce unique mRNA transcripts depending on the cancer type, and RT-PCR can analyze the expression levels of these transcripts.
Technical evolution of RT-PCR
Since the first introduction of Northern blotting in 1977, although the technique has its drawbacks in RNA quantification, such as being time-consuming and requiring large amounts of RNA, RT-PCR has become a popular method for RNA detection and quantification since the invention of PCR by Kary Mullis in 1983. The method of choice for quantitation. This process not only requires no post-processing, but can also measure RNA abundance more than 107 times, further providing quality and quantity data.Currently, RT-PCR technology has developed different operation modes: one-stop and two-step. Two-step RT-PCR requires reverse transcription and PCR amplification to be performed in different test tubes, while one-stop RT-PCR can be completed in one test tube. Although the one-stop method is more convenient for rapid detection, it is susceptible to sample degradation when repeated testing is performed.
Challenges and future directions of RT-PCR
Although RT-PCR technology has many advantages, it still faces some challenges. For example, during multiple cycles of PCR, the exponential growth of complementary DNA (cDNA) after reverse transcription produces inaccurate endpoint quantification, while qRT-PCR overcomes this problem due to its addition of fluorescence monitoring technology. In addition, the high sensitivity means that even trace amounts of DNA contamination can skew the results. Therefore, it is critical to plan and design for sources of variation in the technology.
For example, adding a known amount of RNA as a reference sample can help researchers perform quantitative controls and analyses.
With the continuous evolution of technology, RT-PCR has broad application potential in genetic diagnosis, cancer detection and early screening of diseases. It can be foreseen that in future scientific research, this technology will play a more important role in understanding changes in gene expression and the mechanisms behind them.
With the in-depth study of RT-PCR technology, how will the application of this technology in genetic biology further change our understanding of life processes?
