RT-qPCR Made Simple: A Comprehensive Guide on the Methods, Advantages, Disadvantages, and Everything in Between

Abstract

Introduction: In the world of science, many technologies and methodologies exist to contribute to the process of research. Polymerase Chain Reaction (PCR) is a technology that aids in amplifying specific DNA sequences. PCR can be used to determine the presence of a certain DNA gene. In contrast, reverse transcriptase quantitative PCR (RT-qPCR) converts ribonucleic acids (RNA) into complementary DNA (cDNA) which can then be amplified to give a Ct (threshold cycle) value, a representation of how much of the original RNA transcript was present in the sample. Utility: RT-qPCR is a technique that can be used in many areas of research, including forensic pathology to identify individuals through polymorphic repetitive regions of the DNA called short tandem repeats. This method can also be used in diagnosing various viral diseases such as the recent COVID-19 virus. RT-qPCR is also used in numerous laboratory procedures, such as determining cell growth, cell survival, genetic persistence, and more. Challenges: This method does come with many challenges, such as determining the normalization technique to be used in order to effectively compare the Ct value of the sample with the Ct value of the control gene, as there are numerous ways to perform this comparison. This challenge can be mitigated by establishing a common technique within each lab. Determining which housekeeping gene should be used in the normalization process is also a persistent challenge. This can be addressed by researching the different genes and determining which housekeeping gene will best be established as an accurate control. Ensuring the purification of RNA and gathering knowledge of a few base pairs to design primers are other challenges that must be considered as well but can be resolved fairly easily. Limitations: Limitations such as the difficulty in replication can hinder the reliability of the method. The ‘Monte Carlo’ effect and the lack of an established method for normalization further contribute to the difficulty in comparing studies with differing RT-qPCR protocols used. These limitations can be addressed by publishing data with the exact conditions and methods used in the RT-qPCR reaction.